SAFFRON : science, technology and health 9780128186381, 0128186380, 9780128187401

Table of contents :
Content: Section One: Cultural and Social Aspects of Saffron 1. Saffron and Folklore 2. Saffron and Religions 3. Saffron in the Ancient History of Iran Section Two: Saffron Production 4. Evolution, Botanical and Agricultural Characteristics of Saffron (Crocus sativus L.) and Related Species 5. Soil Conditions for Sustainable Saffron Production 6. Water Requirement of Saffron 7. Saffron Seeds- The Corm 8. Advances in Modeling Saffron Growth and Development at Different Scales 9. Saffron Crop Protection 10. Ecophysiology of Saffron 11. Emerging Innovation in Saffron Section Three: Genetics and Biotechnology of Saffron 12. Molecular Biology and Genetics of Crocus sativus L. 13. Tissue and Cell Culture of Saffron 14. Secondary Metabolites in Saffron Section Four: Saffron Processing 15. Dehydration of Saffron Stigmas 16. Assessment and Monitoring of Saffron Microbiological Criteria 17. Saffron Adulteration Section Five: Economy and Trade of Saffron 18. Saffron Cultivation: An Economic Analysis 19. Saffron Marketing: Challenges and Opportunities 20. Environmental-Economic Analysis of Saffron Production with the Emphasis of Energy, Environmental Impacts and Ecosystem Functions Section Six: Saffron and Health 21. History of Saffron in Medicine 22. Phytochemistry of Saffron 23. Saffron in Traditional Medicine 24. The Effectiveness of Saffron (Crocus sativus L.) on Memory Function, Learning Ability and Epilepsy 25. Antidepressant and Antianxiety Properties of Saffron 26. Saffron (Crocus sativus L.) and its Constituents
their Anti-Inflammatory and Immunomodulatory Effects 27. Cardiovascular Effects of Saffron and its Active Constituents 28. Saffron (Crocus sativus L.) and its Main Constituents, their Effect on Respiratory System 29. Saffron in Metabolic Disorders 30. Available Saffron Formulations and Patents

Citation preview

Saffron: Science, Technology and Health

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Woodhead Publishing Series in Food Science, Technology and Nutrition

Saffron: Science, Technology and Health

Edited by Alireza Koocheki Mohammad Khajeh-Hosseini

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-818638-1 (print) ISBN: 978-0-12-818740-1 (online) For information on all Woodhead Publishing publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Megan R Ball Editorial Project Manager: Laura Okidi Production Project Manager: Debasish Ghosh Cover Designer: Alan Studholme Typeset by MPS Limited, Chennai, India

Contents List of contributors Preface Acknowledgments

xv xix xxi

Section I Cultural and historical aspects of saffron

1

1. Saffron and folklore

3

Mohammad Jafari-Ghanavati and Salman Saket 1.1 Introduction 1.2 Aspects of folklore in planting, growing, and harvesting of saffron 1.3 Applications of saffron in folklore 1.3.1 Saffron and food 1.3.2 Saffron in prayers, charms, and talismans 1.3.3 Other uses of saffron 1.3.4 Popular medicine 1.4 Saffron in popular literature 1.4.1 Do-bayti 1.4.2 Riddles 1.4.3 Stories and humor 1.5 Conclusion References Further reading

2. Saffron and religion

3 4 5 5 6 7 8 9 9 10 10 11 11 13

15

Mansour Motamedi, Fayyaz Gharaei and Salman Saket 2.1 Introduction 15 2.2 Saffron in Indian religions 16 2.2.1 Saffron symbols 16 2.2.2 Saffron in Tantara rites 17 2.2.3 Saffron for laundering Gods 17 2.2.4 Saffron color in India’s flag 17 2.2.5 Saffron, the color of religious costumes 18 2.2.6 Saffron celebrations and festivals 18 2.2.7 Saffron color in Buddhism 19

2.3 Semitic religions 2.3.1 Judaism 2.3.2 Islam 2.4 Conclusion References Further reading

19 19 20 21 21 21

3. Saffron in the ancient history of Iran 23 Askar Bahrami, Sayed-Said Mirmohammadsadegh, Seyyede-Fatemeh Zare-Hoseini, Reza Mousavi-Tabari and Salman Saket 3.1 Introduction 3.2 Saffron in Iran before Islam 3.2.1 Use of saffron before Islam 3.2.2 Use of saffron in ancient Iran 3.3 Saffron in Iran after Islam 3.3.1 Cultivation of saffron 3.3.2 Saffron as a commodity 3.3.3 Applications of saffron in Iranian daily life 3.4 Conclusion References Further reading

Section II Safron production

23 23 24 25 28 28 28 30 32 32 34

35

4. Evolution and botany of saffron (Crocus sativus L.) and allied species 37 Mohammad-Hassan Rashed-Mohassel 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8

Introduction Crocus Contractile roots Corm Iridaceae Saffron evolution and phylogeny Pollination and seed growth Sexual reproduction

37 38 40 41 41 43 46 46

v

vi

Contents

4.9 Cultivated saffron (Crocus sativus L.) 4.10 Wild saffron (C. cartwrightianus Herbert) 4.11 Crocus palasii subsp. Haussknechtii 4.12 Crocus oreocreticus Burtt 4.13 Crocus Thomasii Tenore 4.14 Crocus hadriaticus 4.15 Conclusion Acknowledgment References Further reading

5. Soil conditions for sustainable saffron production

47 51 52 53 54 54 55 56 56 57

59

Hamid Shahandeh 5.1 Introduction 5.2 Soil conditions 5.3 Soil texture 5.4 Soil nutrient content 5.5 Saffron nitrogen requirement 5.6 Nitrogen use efficiency in saffron 5.7 Conclusion References

6. Saffron water requirements

59 60 60 61 62 64 65 65

67

Alireza Koocheki, Hamid-Reza Fallahi and Majid Jami-Al-Ahmadi 6.1 Introduction 6.2 Crop coefficients and potential evapotranspiration 6.3 Irrigation scheduling 6.3.1 Before corm lifting 6.3.2 After corm planting 6.3.3 Preflowering irrigation in autumn 6.3.4 During vegetative growth 6.3.5 Summer irrigation 6.4 Rainfed saffron production 6.5 Factors affecting water requirements 6.6 Irrigation methods 6.7 Water quality 6.7.1 Water quality parameters 6.7.2 Salt stress in saffron 6.8 Water-use efficiency and productivity 6.9 Physiological responses of saffron to water stress 6.10 Beneficial water-related approaches for saffron production 6.11 Saffron response to flooding 6.12 Indigenous irrigation knowledge 6.13 Conclusion References

7. Saffron “seed”, the corm Alireza Koocheki and Seyyed-Mohammad Seyyedi 7.1 Introduction 7.2 Corm botanical criteria 7.2.1 Mother and daughter corms 7.2.2 Main and lateral buds 7.2.3 Root system 7.2.4 Developmental stages and phonological description 7.2.5 Corm nutrient content 7.2.6 Field age effect on corm production 7.3 Agronomical practices 7.3.1 Corm lifting time and storage 7.3.2 Planting time 7.3.3 Planting depth 7.3.4 Row spacing, corm density 7.3.5 Corm size/weight 7.3.6 Corm size classification 7.3.7 Planting beds 7.3.8 Application of hormones 7.4 Corm and climate change 7.5 Conclusion References

8. Ecophysiology of saffron 67 67 70 70 70 71 72 78 80 80 81 82 82 83 83 84 85 87 87 88 88

93

93 93 93 94 96 98 98 101 101 101 103 103 104 104 106 107 112 112 113 113

119

Parviz Rezvani-Moghaddam 8.1 Introduction 8.2 Climatic factors for crop production 8.2.1 Temperature 8.2.2 Precipitation 8.3 Lifecycle 8.3.1 Flowering phase 8.3.2 Vegetative phase 8.3.3 Production of replacement corms 8.3.4 Dormant phase 8.4 Growth parameters 8.4.1 Leaf area index 8.4.2 Crop growth rate 8.4.3 Relative growth rate 8.4.4 Net assimilate rate 8.4.5 Leaf area ratio 8.4.6 Leaf weight ratio 8.4.7 Corms 8.4.8 Whole plant 8.4.9 Source and sink relationship in the growth organs 8.5 Effects of environmental changes on the quality of saffron 8.6 Yield determination

119 120 120 121 122 122 122 122 123 123 125 126 126 130 131 131 133 133 134 134 135

8.7 Conclusion References

135 135

9. Advances in modeling saffron growth and development at different scales 139 Mehdi Nassiri Mahallati 9.1 Introduction 9.2 Statistical models 9.2.1 The basics 9.2.2 Crop-weather models for saffron yield prediction 9.3 Artificial neural networks 9.3.1 Artificial neural networks, an overview 9.3.2 Application of artificial neural networks for saffron 9.4 Application of response surface modeling in saffron production 9.4.1 Statistical background 9.4.2 Central composite designs 9.4.3 Application 9.5 Dynamic simulation models 9.5.1 Advances in development of simulation models for saffron 9.5.2 Radiation-based model for saffron growth 9.6 Modeling saffron development and flowering 9.6.1 Hypothetical model of saffron development 9.6.2 Developmental responses in crop species 9.6.3 Structure of the model 9.6.4 Simulation of saffron response to climate change 9.7 Land suitability and zoning methodology for saffron 9.7.1 Objectives and methods of zoning 9.7.2 Application of zoning schemes to saffron 9.8 Conclusion References Further reading

10. Saffron crop protection

139 140 140 140 143 143 144 146 146 146 147 148 150 152 154 154 155 155 158 161 161 162 163 164 167

169

Mohammad Bazoobandi, Hasan Rahimi and Mahmud-Reza Karimi-Shahri 10.1 Introduction 169 10.2 Weeds 169 10.2.1 Weed and saffron ecophysiology 170 10.2.2 Weed presence in saffron fields and possibility of weed control 170

Contents

vii

10.2.3 Critical period of weed control 10.2.4 Dominant weeds in saffron fields 10.2.5 Weed management 10.3 Pests 10.3.1 Mites (Acari) and Insects (Insecta) 10.4 Pathogens 10.4.1 The fungal agents of saffron corm rot 10.4.2 Fusarium rottings of the saffron corm 10.4.3 Nematodes 10.4.4 The saffron pathogenic bacteria 10.4.5 Saffron viruses 10.5 Conclusion References Further reading

171

11. Mechanization of saffron production

171 174 176 177 182 182 182 183 183 183 184 184 185

187

Mohammad-Hossein Saeidirad 11.1 Introduction 11.1.1 The role of mechanization in agricultural development 11.1.2 Economic advantages of saffron mechanization 11.2 Machines for corm production 11.2.1 Physical properties of saffron corms 11.2.2 Corm digging 11.2.3 Corm sorting 11.3 Tillage 11.3.1 Bed preparation for corm planting 11.3.2 Crust breaking 11.4 Corm planting 11.4.1 Planting patterns 11.4.2 Traditional planting methods 11.4.3 Automatic planting machines 11.5 Harvesting saffron flowers 11.5.1 Traditional method of harvesting saffron flowers 11.5.2 Invented picker machines 11.6 Saffron stigma separation 11.6.1 Physical properties of saffron flowers 11.6.2 Traditional stigma
flower separation method 11.6.3 Invented separators 11.7 Conclusion References

187 187 188 188 188 188 189 189 189 190 191 191 192 192 194 194 195 198 198 198 200 203 204

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Contents

12. Emerging innovation in saffron production

205

Mohammad Khajeh-Hosseini and Farnoush Fallahpour 12.1 Introduction 205 12.2 Why is innovation needed? 206 12.3 Production under controlled environments 207 12.3.1 Soilless beds 207 12.3.2 Growth chambers 208 12.3.3 In vitro cultivation 208 12.4 Forced flowering 209 12.5 Nonconventional breeding techniques 209 12.6 Application of hormones 209 12.7 Production in inadequate climates 210 12.8 Organic production 210 12.9 Mechanization 210 12.10 Smart farming 211 12.11 Saffron as feed additive 211 12.12 Saffron byproducts 212 12.13 e-commerce 212 12.14 Conclusion 213 References 213 Further reading 216

Section III Genetics and biotechnology of saffron 13. Utilizing O-mics technologies for saffron valorization

217 219

Matteo Busconi, Giovanna Soffritti and Jose´-Antonio Ferna´ndez 13.1 Introduction 13.2 Analysis of variation in saffron germplasm 13.3 Epigenetics stability 13.4 Detection of adulteration and DNA-based traceability 13.5 Perspectives Acknowledgements References

14. Tissue and cell culture of saffron

230 230 231 232 232 233 234 235 236 239 241 243 243

15. Molecular biology of Crocus sativus

247

Alireza Seifi and Hajar Shayesteh 15.1 Introduction 15.2 Flower development 15.3 Gametogenesis and interspecific hybridization 15.4 Secondary metabolites 15.4.1 Carotenoid biosynthesis in plants 15.4.2 Carotenoid biosynthesis in saffron 15.4.3 Apocarotenoid biosynthesis 15.4.4 Crocin, crocetin, picrocrocin, and safranal 15.4.5 Genetic regulation of carotenoids biosynthesis 15.5 Production of saffron metabolites in microorganisms 15.6 Saffron
microbe interactions 15.7 Molecular response to abiotic stresses 15.8 Conclusion References

247 247 248 248 249 251 251 252 253 254 255 255 255 256

219 220 222 224 226 227 227

229

Nasrin Moshtaghi 14.1 Introduction 14.2 Tissue culture of monocotyledons

14.3 Micropropagation of saffron 14.3.1 Explant preparation 14.3.2 Propagation 14.3.3 Acclimation 14.4 Callus and cell culture 14.5 Direct organogenesis 14.5.1 Generating adventitious shoots 14.5.2 Microcorm production 14.6 Somatic embryogenesis 14.7 Protoplast culture 14.8 Stigma-like structure 14.9 Conclusion References

229 230

Section IV Saffron processing 16. Bioactive ingredients of saffron: extraction, analysis, applications

259 261

Seid-Mahdi Jafari, Maria Z. Tsimidou, Hamid Rajabi and Anastasia Kyriakoudi 16.1 Introduction 16.1.1 International classification of saffron stigmas 16.1.2 Iranian trade categories

261 262 262

Contents

16.2 Saffron drying methods 16.3 Extraction of saffron bioactive components 16.3.1 Conventional extraction techniques 16.3.2 Novel extraction methods 16.4 Characterization of saffron bioactive compounds 16.4.1 UV-Vis spectrophotometry 16.4.2 High performance liquid chromatography (HPLC) 16.4.3 Gas chromatography-mass spectrometry (GC-MS) 16.4.4 Electronic nose technique 16.5 Applications of saffron bioactive ingredients: from prehistory up to 21st century 16.5.1 Food industry 16.5.2 Pharmaceutical industry 16.5.3 Cosmetics and other sectors 16.6 Conclusion Acknowledgment References

17. Dehydration of saffron stigmas

264 264 266 270 272 272 273 279 279

282 282 283 284 285 285 285

291

Arash Koocheki 17.1 17.2 17.3 17.4 17.5

Introduction Drying methods Traditional methods Artificial drying methods Hybrid photovoltaic
thermal solar dryer 17.6 Infrared thin-layer drying 17.7 Freeze drying 17.8 Microwave drying 17.9 Effects of drying on color, aroma, and taste 17.10 Conclusion References

18. Saffron packaging

291 292 292 292 293 294 295 296 296 297 298

301

Arash Koocheki 18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8

Introduction Saffron packaging Paper and paperboard Aluminum foil Glass Low-density polyethylene High-density polyethylene Modified atmosphere packaging

18.9 Nanosilver composite antimicrobial packaging 18.10 Edible and biodegradable films 18.11 Conclusion References

19. Assessment and monitoring of saffron microbiological criteria

ix

304 304 304 305

307

Elnaz Milani 19.1 Introduction 19.2 Microbial critical point in saffron 19.2.1 Microbiological analysis 19.3 Monitoring harvesting of saffron flowers 19.4 Monitoring transportation of saffron flowers 19.5 Microbial decontamination of saffron by different postharvest processes 19.5.1 Effect of drying processes on microbiological quality of dried stigma 19.5.2 Effect of cold plasma process on microbiological quality of dried stigma 19.5.3 Effect of ozone treatment on microbiological quality of dried stigma 19.5.4 Effect of irradiation treatment on microbiological quality of dried stigma 19.5.5 Infrared irradiation treatment 19.6 Saffron packaging 19.6.1 Modified atmosphere packing 19.7 Effect of antibacterial packaging on microbiological quality of dried stigma 19.8 Effect of Hurdle technology on microbiological quality of dried stigma 19.9 Conclusion References Further reading

20. Saffron adulteration

307 308 308 309 310 310

311

313

313

313 314 315 315 315 316 317 317 320

321

Arash Koocheki and Elnaz Milani 301 302 302 302 302 303 303 303

20.1 Introduction 20.2 Detecting adulteration 20.2.1 Chromatographic techniques 20.2.2 Infrared spectroscopy 20.2.3 1H nuclear magnetic resonance 20.2.4 Molecular techniques 20.2.5 Electronic nose 20.3 Conclusion References

321 321 322 325 328 328 331 332 332

x

Contents

Section V Economy and trade of saffron 21. Economic analysis of saffron production

335 337

Introduction World’s main exporters of saffron Price of saffron Saffron production around the world 21.4.1 Iran 21.4.2 India 21.4.3 Greece 21.4.4 Spain 21.4.5 Afghanistan 21.4.6 Summary 21.5 Production function in economics 21.5.1 Production function of saffron 21.6 Economic productivity of saffron 21.6.1 Water productivity of saffron 21.6.2 Labor productivity of saffron 21.6.3 Land productivity of saffron 21.7 Economic performance of saffron 21.7.1 Return on investment 21.7.2 Ratio of return to cost 21.8 Efficiency 21.8.1 Energy use efficiency 21.9 Economic comparative advantage 21.9.1 Revealed comparative advantage 21.9.2 Policy analysis matrix 21.9.3 Nominal protection coefficient 21.9.4 Effective protection coefficient 21.9.5 The domestic resource cost 21.9.6 Based on unit costs 21.10 Some economic advantages of saffron production 21.11 Conclusion References

22. Saffron marketing: challenges and opportunities

337 337 338 339 340 341 341 342 342 343 343 344 344 345 345 345 346 346 346 346 346 349 349 349 352 352 352 353 353 353 354

357

Hosein Mohammadi and Michael Reed 22.1 Introduction 22.2 Problems of exporting and marketing of saffron 22.3 Marketing concepts in the saffron industry 22.4 Marketing management tasks for saffron marketing

23. Environmental economic analysis of saffron production

364 365

367

Leili Abolhassani, Soroor Khorramdel, Michael Reed and Sayed Saghaian

Naser Shahnoushi, Leili Abolhassani, Vida Kavakebi, Michael Reed and Sayed Saghaian 21.1 21.2 21.3 21.4

22.5 Conclusion References

357 357 360 362

23.1 Introduction 23.2 Estimation of potential environmental impacts by lifecycle assessment 23.2.1 Global warming potential 23.2.2 Acidification potential 23.2.3 Aquatic eutrophication potential 23.2.4 Terrestrial eutrophication potential 23.2.5 Aggregated environmental indicator (EcoX) 23.3 Ecological economic analysis of energy use 23.3.1 Ecological energy indicators 23.3.2 Ecological analysis of energy input 23.3.3 Energy economic indicators 23.4 Carbon footprint 23.5 Ecosystem functions and services 23.5.1 Valuation of the ecosystem function 23.5.2 Ecosystem services and impacts from agriculture 23.5.3 Ecosystem services 23.5.4 Environmental impacts 23.6 Green policy analysis matrix of saffron 23.7 Conclusion References

Section VI Saffron and Health 24. Saffron in Persian traditional medicine

367 367 368 369 369 370 371 372 373 375 378 380 381 382 382 382 384 385 387 387

391 393

Mahdi Yousefi and Khosro Shafaghi 24.1 Introduction 24.2 A short history of using saffron in traditional medicine 24.3 A brief survey on principles of Persian medicine focusing on pharmacological aspects 24.3.1 Temperament 24.3.2 Pharmacotherapy

393 394

394 394 395

Contents

24.3.3 Temperament of drugs and medicines 24.4 Origin and history of the word saffron 24.5 History of saffron usage in Persian civilization 24.6 Medical properties of saffron 24.6.1 Botanical aspects 24.6.2 The nature and matter of saffron 24.6.3 Temperament and general properties 24.6.4 Medicinal uses of saffron 24.6.5 Toxicity and adverse effects 24.6.6 Quality assessment 24.7 Conclusion References

25. Antiinflammatory and immunomodulatory effects of saffron and its derivatives

396 397 397 397 397 397 398 402 403 403 403 404

405

Mohammad-Hossein Boskabady, Zahra Gholamnezhad, Mohammad-Reza Khazdair and Jalil Tavakol-Afshari 25.1 Introduction 25.2 Antiinflammatory effects of saffron and its derivatives 25.2.1 Antiinflammatory effects of saffron extracts 25.2.2 Antiinflammatory effects of saffron petals 25.2.3 Antiinflammatory effects of saffron derivatives 25.3 Immunomodulatory effects of saffron and its derivatives 25.3.1 Immunomodulatory effects of saffron extracts 25.3.2 Immunomodulatory effects of saffron derivatives 25.4 Conclusion References Further reading

405 406 406 407 409 412 414 415 417 417 421

26. Effectiveness of saffron on memory function, learning ability, and epilepsy 423 Hamid-Reza Sadeghnia, Arezoo Rajabian and Seyed-Mahmoud Hosseini 26.1 Introduction 26.2 In vitro and preclinical studies 26.2.1 Memory and learning skills

423 423 423

26.2.2 Oxidative stress 26.2.3 Alzheimer’s disease 26.2.4 Seizure 26.3 Clinical studies 26.4 Conclusion References

27. Antidepressant and antianxiety properties of saffron

xi

425 426 427 427 427 428

431

Seyed Ahmad Mohajeri, Samaneh Sepahi and Adel Ghorani Azam 27.1 Introduction 27.2 Nervous system 27.3 Depression and anxiety 27.3.1 Pathophysiology of depression and anxiety 27.3.2 Epidemiology of depression and anxiety 27.3.3 Causes of depression and anxiety 27.4 Antidepressants 27.4.1 Classification of antidepressants and antianxiety drugs 27.4.2 Pharmacokinetics 27.4.3 Mechanism of action of antidepressants and antianxiety drugs 27.4.4 Possible side effects of antidepressants 27.5 Traditional medicine for the treatment of depression and anxiety 27.6 Saffron 27.6.1 Chemical compounds of saffron 27.6.2 Pharmaceutical applications of saffron in traditional medicine 27.6.3 Pharmacology of saffron and its active ingredients 27.6.4 In vivo studies on the effects of saffron and its active compounds on depression and anxiety 27.6.5 Antidepressant and anxiolytic properties of saffron in clinical practice 27.6.6 Mechanistic pathway for antidepressant and antianxiety effects 27.6.7 Effect of saffron and saffron compounds on nervous system diseases

431 431 432 432 432 433 433 433 434

434 434 435 435 435

436 436

436

437

438

438

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Contents

27.7 Conclusion References

28. Application of saffron as a neuroprotective agent

440 441

445

Shahin Akhondzadeh, Seyyed-Hosein Mortazavi, Erfan Sahebolzamani and Amirhosein Mortezaei 28.1 Alzheimer’s disease 28.1.1 Introduction and epidemiology 28.1.2 Signs and symptoms 28.1.3 Pathogenesis 28.1.4 Pharmacological therapy 28.2 Saffron in Alzheimer’s disease treatment 28.2.1 Introduction to saffron 28.2.2 Effects of saffron on the central nervous system 28.2.3 Neuroprotective activity of saffron 28.2.4 Saffron clinical trials on Alzheimer’s disease 28.3 Other herbal medicines and Alzheimer’s disease 28.4 Conclusion References

29. Cardiovascular effects of saffron and its active constituents

445 445 445 445 446 446 446 446 448

30. Saffron, its main derivatives, and their effects on the respiratory system

449 449 449

451 451 452 452

462

462

463 464 464

466 467 467

31. Saffron’s role in metabolic disorders

471

Ahmad Ghorbani 31.1 Introduction 31.2 Effects of saffron on obesity 31.3 Effects of saffron on diabetes 31.3.1 Antihyperglycemic effects of saffron 31.3.2 Antihyperglycemic mechanisms of saffron 31.3.3 Effects of saffron on diabetic complications 31.4 Conclusion References

471 471 472 473 473 476 479 480

453 453 454

455 457 457

461

Mohammad-Hossein Boskabady, Vahideh Ghorani and Azam Alavinezhad 30.1 Introduction

462

448

Bibi-Marjan Razavi and Hossein Hosseinzadeh 29.1 Introduction 29.2 Cardiovascular pharmacological effects of saffron 29.2.1 Antiarrhythmic and antiischemic effects 29.2.2 Protective effects against cardiac hypertrophy 29.2.3 Effects of saffron and its active constituents on blood pressure 29.2.4 Antiatherosclerotic effects of saffron and its active constituents 29.2.5 Protective effects of saffron and its active constituents on natural and chemical toxins 29.3 Conclusion References

30.2 Bronchodilatory effect of saffron and its derivatives 30.2.1 Relaxant effect of saffron extracts on airway smooth muscle 30.2.2 Relaxant effect of saffron derivatives on airway smooth muscle 30.2.3 Possible mechanisms of the airway smooth muscle relaxant effect of saffron and its derivatives 30.3 Prophylactic effect of saffron and its derivatives on respiratory disorders 30.3.1 Prophylactic effect of saffron extracts on respiratory disorders 30.3.2 Prophylactic effect of saffron derivatives on respiratory disorders 30.4 Conclusion References

461

32. Anticancer properties of saffron

485

Jalil Tavakol-Afshari, Mohammad-Hossein Boskabady and Roshanak Salari 32.1 Introduction 32.2 Lung cancer 32.3 Pancreatic cancer 32.4 Colorectal cancer 32.5 Breast cancer 32.6 Leukemia 32.7 Hepatic cancer 32.8 Cervical cancer 32.9 Skin cancer 32.10 Prostate cancer 32.11 Gastric cancer 32.12 Conclusion References

485 486 487 487 487 488 489 489 490 490 490 491 491

Contents

33. Available saffron formulations and product patents

493

Seyed Ahmad Mohajeri, Narges Hedayati and Mehri Bemani-Naeini 33.1 Introduction 33.2 Skin care products 33.2.1 Creams 33.2.2 Masks 33.3 Health care products 33.3.1 Toothpaste 33.3.2 Drugs for kidney health 33.3.3 Compositions containing enriched natural crocin and/or crocetin 33.3.4 Hair conditioner 33.4 Therapeutic products 33.4.1 Chinese medicine for treatment of angiitis 33.4.2 Krocina tablet 33.4.3 Topical spray 33.4.4 Arthritis and rheumatism liquid patch 33.4.5 Ointment for treating dermatitis 33.4.6 Antiacne ointment 33.4.7 Topical spray for treating skin scars 33.4.8 Oral compositions 33.4.9 Tibetan nighttime medicine 33.4.10 Antimicrobial composition 33.4.11 Chinese medicinal composition 33.4.12 Gout medicine 33.4.13 Prepregnancy fetus protection pills 33.4.14 Cold and flu symptomatic relief composition 33.4.15 Herbal composition for treating diabetes 33.4.16 Multiglycosides saffron tablets 33.4.17 Externally applied Chinese medicine 33.4.18 Satiation agent for the treatment of obesity 33.4.19 Medicine for treating chronic obstructive pulmonary disease 33.4.20 Topical treatment for rheumatic arthritis 33.4.21 Chinese medicine for treatment and prevention of rheumatic arthritis 33.4.22 Saffron-based compositions for treating of duodenal bulbar ulcer or inflammation

494 494 494 495 496 496 496

496 496 496 496 496 497 497 497 497 497 497 497 498 498 498 498 498 498 498 499 499 499 499

499

499

33.4.23 Traditional Chinese medicine for treating gastric ulcer 33.4.24 Topical treatment for breast cancer 33.4.25 Chinese medicine for treating brain apoplexy 33.4.26 Saffrotin capsule 33.4.27 Composition for the treatment and prevention of degenerative eye disorders 33.4.28 Medicine for treating prostatic hyperplasia 33.4.29 Chinese medicine for treatment of gynecologic diseases 33.4.30 Traditional Chinese composition for treating dysmenorrhea 33.4.31 Composition for treating endocrine dysfunction 33.4.32 Chinese formulation for treating premature ovarian failure 33.4.33 Traditional Chinese medicine for treating rheumatic heart disease 33.4.34 Chinese medicine for treating cataracts 33.4.35 Traditional Chinese medicine capable of treating cervical spondylosis 33.4.36 Traditional Chinese medicine preparation for treating qi stagnation and blood stasis 33.4.37 Chinese medicine for treating osteoproliferation and herniated disk 33.4.38 Traditional Chinese medicine for treating blocking antibody deficiency in recurrent spontaneous abortion 33.4.39 Herbal medicine formula for treating nasopharyngitis 33.4.40 Chinese medicine for treating lung tumor 33.4.41 Medicine for treating damp-heat stagnation (abdominal mass) 33.4.42 Traditional Chinese medicine composition for treating ascites due to cirrhosis 33.4.43 Traditional Chinese medicine for treating nonulcer dyspepsia 33.4.44 Chinese medicine for treating peptic ulcer

xiii

499 500 500 500

500 500 500

501 501

501

501 501

501

502

502

502 502 502 502

503 503 503

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33.4.45 Medicine for treating ankylosing spondylitis 33.4.46 Externally-applied wet tissue for treating measles 33.4.47 Treatment of herpes zoster 33.4.48 Chinese composition for treating septic shock 33.4.49 Drug for treating bone injury 33.4.50 Traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases 33.4.51 Medicine for treating eyelid eczema 33.5 Food products 33.5.1 Vegetable drinks 33.5.2 Healthy drink prepared from saffron pollen 33.6 Conclusion References

503 503 503 504 504

504 504 504 504 504 513 513

34. Safety and toxicity of saffron

517

Soghra Mehri, Bibi-Marjan Razavi and Hossein Hosseinzadeh 34.1 Introduction 34.2 Experimental data on the safety and toxicity of saffron and its bioactive ingredients in animal models 34.2.1 Acute toxicity 34.2.2 Subacute toxicity 34.2.3 Subchronic and chronic toxicity 34.2.4 Developmental toxicity 34.2.5 Mutagenicity and genotoxicity 34.3 Clinical studies on safe and toxic doses of saffron and its bioactive ingredients 34.4 Conclusion References Index

517

518 518 518 519 520 521 523 527 528 531

List of contributors Leili Abolhassani Department of Agricultural Economics, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Shahin Akhondzadeh Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences, Tehran, Iran Azam Alavinezhad Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Zahra Gholamnezhad Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Vahideh Ghorani Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Fundation,

Adel Ghorani Azam Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Mohammad Bazoobandi Department of Plant Pests and Diseases Protection, Khorasan Razavi Agricultural and Natural Resources Research Center, Mashhad, Iran

Ahmad Ghorbani Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran

Mehri Bemani-Naeini Nanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Narges Hedayati Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Mohammad-Hossein Boskabady Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Seyed-Mahmoud Hosseini Division of Neurocognitive Sciences, Psychiatry and Behavioral Sciences Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Askar Bahrami Encyclopedia Tehran, Iran

Islamica

Matteo Busconi Department of Sustainable Crop Production, Faculty of Agriculture, Food and Environmental Sciences, Catholic University of the Sacred Heart, Piacenza, Italy Hamid-Reza Fallahi Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Birjand, Birjand, Iran Farnoush Fallahpour Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Hossein Hosseinzadeh Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Seid-Mahdi Jafari Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran Mohammad Jafari-Ghanavati Center for the Great Islamic Encyclopedia, Tehran, Iran

Jose´-Antonio Ferna´ndez Laboratory of Biotechnology and Natural Resources, IDR/ETSIAM, University of Castilla
La Mancha, Albacete, Spain

Majid Jami-Al-Ahmadi Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Birjand, Birjand, Iran

Fayyaz Gharaei Department of Religion and Comparative Mysticism, Faculty of Theology and Islamic Studies, Ferdowsi University of Mashhad, Mashhad, Iran

Mahmud-Reza Karimi-Shahri Department of Plant Pests and Diseases Protection, Khorasan Razavi Agricultural and Natural Resources Research Center, Mashhad, Iran xv

xvi

List of contributors

Vida Kavakebi Department of Agricultural Economics, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Mansour Motamedi Department of Religion and Comparative Mysticism, Faculty of Theology and Islamic Studies, Ferdowsi University of Mashhad, Mashhad, Iran

Mohammad Khajeh-Hosseini Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Reza Mousavi-Tabari Written Center, Tehran, Iran

Mohammad-Reza Khazdair Cardiovascular Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran

Mehdi Nassiri Mahallati Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Soroor Khorramdel Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Hasan Rahimi Department of Plant Pests and Diseases Protection, Khorasan Razavi Agricultural and Natural Resources Research Center, Mashhad, Iran

Alireza Koocheki Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Hamid Rajabi Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

Arash Koocheki Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Anastasia Kyriakoudi Laboratory of Food Chemistry and Technology (LFCT), School of Chemistry, Aristotle University of Thessaloniki (AUTH), Thessaloniki, Greece Soghra Mehri Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Elnaz Milani Food Science and Technology Research Institute, ACECR, Mashhad, Iran Sayed-Said Mirmohammadsadegh Written Research Center, Tehran, Iran

Heritage

Seyed Ahmad Mohajeri Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Hosein Mohammadi Department of Agricultural Economics, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Seyyed-Hosein Mortazavi Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences, Tehran, Iran Amirhosein Mortezaei Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences, Tehran, Iran Nasrin Moshtaghi Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Heritage

Research

Arezoo Rajabian Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran Mohammad-Hassan Rashed-Mohassel Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Bibi-Marjan Razavi Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Michael Reed Department of Agricultural Economics, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, United States Parviz Rezvani-Moghaddam Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Hamid-Reza Sadeghnia Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Division of Neurocognitive Sciences, Psychiatry and Behavioral Sciences Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Mohammad-Hossein Saeidirad Department of Agricultural Engineering Research, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran Sayed Saghaian Department of Agricultural Economics, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, United States

List of contributors

xvii

Erfan Sahebolzamani Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences, Tehran, Iran

Naser Shahnoushi Department of Agricultural Economics, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Salman Saket Department of Persian Language and Literature, Faculty of Letters and Humanities, Ferdowsi University of Mashhad, Mashhad, Iran

Hajar Shayesteh Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Roshanak Salari Department of Pharmaceutical Sciences in Persian Medicine, School of Persian and Complementary Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Giovanna Soffritti Department of Sustainable Crop Production, Faculty of Agriculture, Food and Environmental Sciences, Catholic University of the Sacred Heart, Piacenza, Italy

Alireza Seifi Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Jalil Tavakol-Afshari Immunology Research Group, Buali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran

Samaneh Sepahi Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

Maria Z. Tsimidou Laboratory of Food Chemistry and Technology (LFCT), School of Chemistry, Aristotle University of Thessaloniki (AUTH), Thessaloniki, Greece

Seyyed-Mohammad Seyyedi Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Khosro Shafaghi Department of Nutrition and Biochemistry, Faculty of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran Hamid Shahandeh Texas A&M University, College Station, TX, United States

Mahdi Yousefi Department of Persian Medicine, School of Persian and Complementary Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Seyyede-Fatemeh Zare-Hoseini Department of Religion and Comparative Mysticism, Faculty of Theology and Islamic Studies, Ferdowsi University of Mashhad, Mashhad, Iran

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Preface Saffron as the world’s most expensive agricultural commodity is also one of the most fascinating plants, not only due to its economic value but also because of the mysteries surrounding its origin and domestication that has ties with myths and legends. Though the origin, region, and time of saffron domestication has been a matter of discrepancy and argument, its history goes back to the Sumerian and Babylonian era up to 3000 BCE and has been grown in the Near East and Mediterranean basin since the late Bronze age. Iran, Greece, Kashmir, and Turkey have been reported as the possible site of its domestication and cultivation in the past. Although it is a niche cash crop and not many countries in the world produces this valuable spice, due to its four exceptional quality attributes—aroma, flavor, taste, and color—it is known worldwide as an elegant and charming food item. Saffron as a crop with a miniature form that is processed by hand, resembles a “handmade” item and has been used as a condiment in the cuisines of kings and khalifs, as a medicine to cure maladies and disorders in traditional medicine, as a dye for elegant gowns of queens and celebrities, as ink for writing old inscription, and today as a promising and novel biomedical substance with a wide range of cytotoxic, anticarcinogenic, and antitumor properties. Saffron possesses a set of somewhat unique ecological, physiological, and agronomic properties along with strong socioeconomical criteria most suitable for small-scale family farming and community-supported agricultural systems. This spice inspires family farmers and local communities and provides a livelihood for them. Women’s contribution to saffron production, particularly in harvesting and postharvest processing, is also significant. It represents a valuable natural and cultural heritage based on the experiences accumulated by indigenous knowledge of local farming communities. Saffron production and processing are tied to the collective, cooperative, and collaborative organizations built by farmers, families, neighbors, and other members of communities, hence strengthening social, economic, and cultural interdependencies of rural areas. This crop has potential in terms of income and job generation, resource-use efficiency and productivity, and environmental friendliness, creating harmony between humans and nature through ecosystem balance and esthetic beauty. Today the total production of saffron worldwide is more than 400 tons per year from around 110,000 ha of land with a mean yield of nearly 3.6 kg/ha, which is relatively low, despite gradual introduction of conventional chemical inputs to saffron cropping areas. This could be mainly due to lack of proper farm management practices and postharvest losses along with global environmental changes. Long-term analysis of yield gap in Iran indicates a potential yield of 10 kg/ha under present environmental conditions in saffron growing areas. However, labor shortage and high expenses for harvesting and separation of stigmas from the flowers are current challenges. As older generations of farmers are retiring, younger farmers are replacing their techniques with innovative scientific and technical approaches focused on production and processing. This will impact present sustainable and resilient farming systems and jeopardize practices evolved through indigenous knowledge for thousands of years and hence put pressure on small-scale family and community-supported saffron production systems. However, application of new technologies in saffron production seems to be inevitable in the near future due to the cost of production and international market demands. The problem of adulteration is also a challenge facing international markets particularly when saffron is used as a medicine. This spice has been the most adulterated food commodity in the world throughout history and requires international rules and regulations to enforce strict standards to ensure quality and originality. Development of simple and easy-to-handle devices for quality control and detection of adulterated products will enhance this process. Furthermore, the marketing and economics of saffron by its main producers must be addressed. For example, it is believed that over 80% of Iranian saffron is reexported to third-party countries. Since saffron is usually exported in bulk form, it is easily packed and relabeled. Control of smuggling and adoption of branding can help reduce the chance of adulteration. Market access for small holders in producing countries is another challenge that must be addressed. A sound business model to initiate market networks to facilitate direct communication between producers and consumers through social media and the internet is one way to address this challenge. In this way, market channels will be diversified and the benefits reach producers and consumers alike. In terms of consumption and use of saffron as food in different forms, xix

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Preface

as a cosmetic component and as a biomedical item, quality control and determination of saffron constituents based on new technologies and novel standards is key issue to meeting the main objectives of the production-consumption chain of saffron. This is important when saffron is used in biomedical applications associated with health and medical remedies. This book addresses, in a holistic nature, all aspects of saffron production including sociocultural, economical, environmental, biomedical, and dietary issues in 34 chapters. Scientists from a wide range of disciplines contributed to the text, making it a comprehensive book for anyone involved with saffron production including consumers, companies, scientists, and students. Editors Alireza Koocheki and Mohammad Khajeh-Hosseini

Acknowledgments We would like to thank all of the people who helped fulfill our long-lasting desire to complete this comprehensive book on saffron, including our colleagues and coordinators who organized various sections and AREEO for their support and encouragement.

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Section I

Cultural and historical aspects of saffron

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Chapter 1

Saffron and folklore Mohammad Jafari-Ghanavati1 and Salman Saket2 1

Center for the Great Islamic Encyclopedia, Tehran, Iran, 2Department of Persian Language and Literature, Faculty of Letters and Humanities,

Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 1.1 Introduction 1.2 Aspects of folklore in planting, growing, and harvesting of saffron 1.3 Applications of saffron in folklore 1.3.1 Saffron and food 1.3.2 Saffron in prayers, charms, and talismans 1.3.3 Other uses of saffron 1.3.4 Popular medicine

1.1

3 4 5 5 6 7 8

1.4 Saffron in popular literature 1.4.1 Do-bayti 1.4.2 Riddles 1.4.3 Stories and humor 1.5 Conclusion References Further reading

9 9 10 10 11 11 13

Introduction

The word Crocus comes from Greek mythology. It is said that Crocus, one of the friends of the god Hermes, was hurt by him during a disc throw. Three drops of blood from his head fell down on the center of a saffron flower and thus the three stigmas of the saffron flower grew and were named Crocus (Kakisis, 2017, p. 1). In another story there was a boy named Crocus, who adored Hermes, and after he died, was transformed him into a saffron flower by Hermes (Magdalini, 2017, p. 9). Later on during the era of scientific naming of plants, Crocus was chosen as the name of genus of flowering plants which saffron belongs to. Perhaps the genus name of many other plants also root in the popular culture. Popular culture refers to all aspects of informal culture, whether material or immaterial, including aspects of everyday life. Saffron has long been used in popular culture, in particular in rituals and ceremonies particularly in the regions of the world where saffron is produced for a long time. For example, in the text Materia Medica, written by a Greek medical practitioner named Pedanio Dioscorides, the use of saffron is suggested for its healing properties (Christodoulou et al., 2015). In Kashmir people boil the bulbs of saffron in cow’s milk and use the resulting paste for joint inflammation. Saffron in milk is also used by women to improve the skin color of their newborns (Srivastava et al., 1985, p. 72). In addition to therapeutic practices, saffron is used in meals, festivals, gifts, and in social interactions as in the Spanish culture (Halvorson, 2008, p. 24). The tradition of saffron cultivation in La Mancha in Spain is a part of the local folklore, represented in songs and stories such as in the opera The Rose of the Saffron. It is also customary in La Mancha to give couples a few filaments of saffron to wish them a prosperous future (Azafranes Manchegos, 2019). In Switzerland, meadow saffron flower is attached to the neck of infants to ward off illness (Daniels and Stevans, 2003, vol. 2, p. 841). In India, Pakistan, and Malaysia rice colored yellow with saffron or turmeric is widely used. Kashmir washermen also used saffron on clothing as an insect repellant (Hutchings, 2004, p. 61). As most saffron planting took place in Iran historically, it is obvious that most of the folkloric related aspects are seen in Iran. As in other cultures, saffron is a part of popular culture, and is reflected in oral literature.

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00001-0 © 2020 Elsevier Inc. All rights reserved.

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SECTION | I Cultural and historical aspects of saffron

1.2

Aspects of folklore in planting, growing, and harvesting of saffron

The cultivation of saffron corms, like all other agricultural activities, involves special traditions and practices. For example, in Kashmar, a member of the family makes a fire on the ground and then puts some of the fire on a plate or small tray, and someone else puts some sweets, or raisins and almonds on the other plate . Then both of them alongside of the all members of the family and also workers go to the farm. Wild rue (esfand), sometimes mixed with salt, is poured on the fire. As the smoke of the wild rue rises, one person who has a nice and loud voice sings this song: Wild rue is green/wild rue creates greenness. Hundreds of tasks can be done with wild rue/the Prophet (Mohammad) burnt wild rue to ward off harm. Wild rue is immunity from evil eye of the people, send blessings on the bright shrine of the Prophet.

During the ceremony the audience send blessings (salawat) to the Prophet Mohammad three times and then eat the sweets and raisins. Then they say Bismillah Al-Rahman al-Rahim. When the workers are working, they also sing (or shout) do-bayti (two-couplet) to removal tiredness. This is one example of a do-bayti sung during work: I cried on a mountain-top the name of Ali, the Lion of God, O Ali, the Lion of God, the King of Men, make our sad hearts happy. O God, I have a dear travelling, and I yearn to reunite with them, Everyone calms me by saying the departed is coming, but I have no news nor messenger for that. The star rose and we rose afterwards, O God, when will the caravan starts its journey, O God, I wish the caravan stayed here one more night, as I have a journey ahead and a heart stuck behind.

The harvesting of the saffron flowers is also accompanied by rituals. In many areas, they first smoke wild rue, and in some areas, such as the village of Kakhuk, Birjand, they also burn boswellia. Then they form a group and curse Satan and “the evil eye,” and begin with salawat and bismillah. In some areas, someone sings the following song while others shout “bismillah” at the same time: Name of God/Bismillah Problem solver/Bismillah Kindness and purity/Bismillah Beginning of work/Bismillah End of work/Bismillah Away from calamity/Bismillah When worshiping/Bismillah Our words/Bismillah Thanks God/Bismillah In some areas, such as Kashmar, if the land is large enough and there are a lot of saffron flowers a sheep is sacrificed to ward off the evil eye. Before the sacrifice, they move the sheep once around the land. During the flower harvesting, they also sing do-baytis to removal tiredness. Sometimes people sing this song to Ali (a significant figure for Shia Muslims) while others call out Ali’s name: Conqueror of Khaybar/Ali Companion of Prophet/Ali Warrior man/Ali Enemy of oppressors/Ali Companion of Quran/Ali Najah is the bottom part of the stigma of saffron flower. The upper part of the stigma has three red branches. Some stigmas have more than three branches. Stigmas with six branches, called “shishtaki,” and are considered a good omen, and they will sacrifice a sheep in the same place. During the sacrifice, the blood of the sheep is poured on to the soil of the land where saffron with shishtaki grown. In some areas, such as the village of Khoorzad, Ferdows, landowners offer prizes for flowers with stigma more than three branches. After the flowers are picked, the stigmas are separated from the petals (or according to the people of Ferdows, separating of petals from stigmas called “pare gol”). In some areas, this practice of separating petals from stigmas is called “gol par kerd” or “gol wa kardan.” If a stigma found with

Saffron and folklore Chapter | 1

5

twisted branches (Sargols), it will be considered a good omen that this year saffron will become expensive. The separation of stigmas and petals is mainly done by women and girls, who read do-baytis and narrate stories while they work. Tea, melons, and dates are often served. In Ghaen, it is customary for a young man to help his fiance´e’s family by participating in the stigma separation ceremony for at least one night. In some areas, mice eat the saffron corms and cause problems for landowners. Smoke is often used to get rid of them. Hamedani in the seventh century referred to the mouse pest and describes the way the people addressed it (Sotoudeh, 1989): “The peasants have an iron nail which they put in land to find out where the mouse canals are. After that, the hole of the mouse will be examined and sometimes they smoke into the openings of the holes to see from where the smoke comes out . . . as smoke comes out, they know the size of the hole and the boundaries of the hole and find out where it has given birth to its off springs. Then they find the mouse and kill it.” Furthermore, prizes were given to workers who found and destroyed mice holes in the fields (Sotoudeh, 1989, pp. 204, 205). After 300 years, the author of Ershad al-Zira’ah (written by Abunasri Heravi) also recommended the same method (Moshiri, 1967, p. 211). Today, motorcycle exhaust smoke is put into mouse holes using rubber tubes to remove mice.

1.3

Applications of saffron in folklore

1.3.1 Saffron and food Saffron has long been known as a material in the Iranian cooking systems. For example, in the story of Zahak (devilish creature in the Iranian epic narratives), while describing the foods Iblis (specific name for devil) prepared for Zahak daily: On the fourth day, he set a long table on which meat stew of the back of calf was cooked in saffron and rosewater, as well as old wine and fine musk.

Zahak was greatly amazed: As soon as Zahak reached for the food and ate some, he was taken aback by the taste.

He tells Iblis that he will fulfill any wish he desires (Khaleghi-Motlagh, 2007, vol. 1, pp. 49, 50). The use of saffron in food is also attributed to Jamshid (Indo-Iranian mythological hero) (Hasouri, 2005, p. 17). Saffron was commonly used in the Achaemenid and Sassanian periods (see Abrishami, 2004, pp. 241
252) and continued in the Islamic period. Many poets and scholars have described foods such as saffron Zirbai (refer to NezamiGanjavi, 1995, p. 275; Shafiee-Kadkani, 2005, pp. 98, 99, 120); various types of rice saffron (Afshar, 2005, various pages); chickpea dishes (nokhodab) (Fassaei, 2003, p. 8); and saffron samosa (Golchin-Maani, 1967, p. 414, for more details see Abrishami, 2004, pp. 268
335). The use of saffron, especially as a seasoning, was so common during the Safavid era that, until beginning of the current century, one of the dowry items of the bridegroom was a pestle used for grounding saffron (Shahri, 2004, vol. 3, p. 183). Today, in different regions of Iran, saffron is used as a seasoning in many foods, including various types of rice, halva, sweets, breads, stews, and sirups (see Daryabandari, 2006, throughout the book; Khavar, 2009, throughout the book; Moayyed-Mohseni, 2002, different pages). Saffron is required in some foods and sirups for certain rituals and ceremonies. In Tehran, hundreds of years ago, one of the sirups placed on the bride and groom’s bed was sirup made of sugar, rosewater, and saffron (Saadvandian, 2010, p. 50). In Sarvestan, Fars province, one of the foods sent from the bridegroom’s family to the bride’s home on the day before the wedding is saffron (Homayouni, 1992, p. 498). In Lapuie, Fars province, items needed by the bride and groom during the first few days or marriage, including saffron, are placed in the bridal chamber (Jafari and Jafari, 2007, vol. 2, p. 187). In old Tehran, the wedding night dinner was usually sweetened pilaf with abundant saffron. This meal was also served during bridal showers (Katiraei, 1969, pp. 220, 237). In old Tehran, it was customary for the family of the bride to prepare and send saffron halva for the bride and groom on the day of the bridal shower (Mostowfi, 1992, vol. 1, p. 350). In some families, the bride’s mother also sent rice flour, oil, and saffron to the groom’s house where they would cook halva (Shahri, 2004, vol. 3, p. 139). In Sarvestan, after removing the bridal chamber, which takes place a week after the wedding night, the mother of the bride sends a special halva, one of the ingredients of which is saffron (Homayouni, 1992, p. 59). In Ardakan, in Yazd province, the halva is cooked in the bridegroom’s house one day after the wedding, and it is called “tarhalva” (Tabatabei-Ardakani, 2001, p. 352). In Tehran and other cities, one of the items in the layette is saffron (Homayouni, 1992, p. 425; Jafari and Jafari, 2007, vol. 1, p. 111; Shahri, 2004, vol. 3, p. 250; Shahri, 1999).

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SECTION | I Cultural and historical aspects of saffron

In the port of Mahshahr, Khuzestan province, Hendijan, Khuzestan province, and some of the villages in the area, people hold the first Eid of the deceased on Eid al-Fitr. At the ceremony, participants drink brewed saffron. One of the votive offerings in Khorasan is an Omaj Komaj Ash dish that is cooked for the fulfillment of any wish and is made with saffron (see Shakurzadeh, 1984, pp. 28, 29). In this part of Iran, if a woman has trouble delivering a baby or has a history of abortion, halva is cooked with saffron and given as a votive offering; it is called Halva of the Twelve Imams (Shakurzadeh, 1984, p. 36). In many parts of Iran, cookies are served at various celebrations, including Nowruz. For example, in Khor and Biabanak, Isfahan province, a confection called Chang Mal (a mixture of lavash, samanou, dates, animal oil, saffron, sesame) is baked and served on a Nowruz table (Hekmat-Yaghmaei, 2013, p. 252). In Birjand, a bread made with saffron called “khoshki” is served at Nowruz meetings and gatherings (Rezaei, 2008, p. 364; Shakurzadeh, 1984, p. 105). In some parts of Iran, saffron is used in Ramadan specialty foods. In old Tehran, women purchased saffron in addition to other food for pre-Iftar or Iftar desserts in preparation for Ramadan. Elderly women also smashed opium seed with saffron, marinated it with rosewater, rolled the mixture into small balls before the beginning of Ramadan, and then ate one or two of them before sunrise; they believed it gave them great strength (Saadvandian, 2010, p. 150). The people of Tehran also added saffron to some of their Iftar foods, including Ajil Abgoosht, taskabab, shami dumplings, and potato casserole (Shahri, 2004, pp. 313
323). The people of Arak, the capital of Markazi province, also believe that during Ramadan the stomach absorbs water and so they eat a food called Zirehjoosh, which is fatty and sweet and has saffron in it (Vakilian, 2001, pp. 191, 192). Among other things, saffron sirup is used as a votive offering, especially during the period of Muharram. To prepare it, first sugar is dissolved in water and then saffron that they have separately brewed is added, while sending blessing to the household of the prophet (salawat), then a few grains of cardamom are added for consistency. After cooling, cold water and ice are added and the tea is distributed among the mourners. In Talesh, Gilan province, sesame is also added to the tea. In Hamedan, the capital of Hamedan province, this sirup along with apple Faloodeh is served at baby showers. In Kilan, Tehran province, four cups of this sirup are placed on the bridegroom’s bed; one cup for the bridegroom, two cups for the groomsmen, and one cup as the prize for the winner of gush bekandag game (lit.: ear twisting). During this game, the groom’s father tosses a coin at the start of the game and whoever takes the coin, has to pass a tough test, when his ear is strongly twisted until he could name seven elderly widows from the neighbors. If he manages to give all seven names, he gets drink the saffron sirup and receives a valuable prize from the groom (Anizadeh, n.d., pp. 226, 227). In Bushehr, Genaveh, and in the neighborhoods of these cities, sugar candy is used instead of sugar in the sirup, and this rosewater candy is consumed during Iftar. In Iran, common dishes at funerals and anniversaries, as well as on Fridays for votive offerings, are starchy halva or flour and rice halva with saffron. For example, saffron is one of the seasonings in Sholehzard, one of the dishes served as a votive offering. In Ashrafieh, Gilan province, saffron cookies called Debij cakes are only served during the month of Muharram (Faghih-Mohammadi Jalali, 2003, p. 123).

1.3.2 Saffron in prayers, charms, and talismans In Iranian culture and civilization, saffron is used in a variety of prayers, charms, and talismans. For example, Zoroastrians wrote in Pahlavi script on a deer’s skin or paper and put it on the doors of their houses (Saberi-Eftekhari, 2014, p. 680). In the narratives of Darab Hormozdyar a charm is also used to relieve headaches. The person who writes this charm must first read the remembrance (bazh) the God of Ordibehesht, ground and combine musk and saffron with wine, and then write the charm with the solution obtained on a deer skin and attach it to the left arm (Unwala, 1922, vol. 2, p. 275). This kind of charm is also common among Muslims in Iran. For example, in Borujerd, Lorestan province, a prayer with the essence of saffron is written and attached to the patient’s arm for healing (Izadi et al., 1994, p. 147). In old Tehran, it was customary for a new mother to wrap the “shirt of Quran” around her neck after giving birth to “ward off danger.” The shirt of Quran was made of calico pieces on which the words of the Quran were written in small script with saffron and rosewater; if the shirt of Quran was not available, Yasin Ghaleh was used (Saadvandian, 2010, pp. 84, 85). Yasin Ghaleh (or Yasin Halgheh) is a piece of white cloth on which Yasin (the 36th sura (chapter) of Quran) is written on it with musk or saffron and making a hole in the middle of the cloth; and then people pass through it with the intention of solving a specific problem (Asadian-Khorramabadi et al., 1979, p. 173). In Golbaf, Kerman province, Ayat al-korsi is written with saffron on a clean dress that is put in water to allow the words to dissolve. Then the water is drunk for healing (Asadi-Kougi, 2000, p. 287; for other samples of curing disease, see Boshra, 2010, p. 81). In Khorasan, if the bridegroom had an impotency issue at consummation night, a prayer

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beginning with the phrase “Enna Fatahna” was read and blow it on some preprepared candy, rosewater, and saffron. After that, they dissolved these three substances in water. Half of the solution was poured on the groom and the other half was given to him to drink (Shakurzadeh, 1984, p. 620). In some books on occult sciences, prayers to attract loved ones or to keep enemies away are written with saffron or saffron and musk (see Ghaem Magham Farahani, 1987, pp. 403
425; Kashefi-Sabzevari, n.d., pp. 132
140). Some of these rituals are reflected in myths in which the prayers are described by amulet writers who write with saffron on paper (e.g., see Anjavi-Shirazi, 2015, p. 312; Sarmad, 2003, pp. 57
60). Saffron is also used for prayers and rituals related to money. In Shahroud, Semnan province, before the turn of the new year, people gather in old mosques and tekyehs and write the seven verses of the Quran that begin with salam with saffron on the back of porcelain plate and bowls. This continues until the new year. Then the bowls and plates are dipped in water, and the water is drunk. Part of the water is drunk by family members and the other part used in food preparation (Shariatzadeh, 1992, p. 420). This prayer, better known as the seven salams, is also common in Central Asia where the “seven salams” are written with saffron on paper that is put it in apricot juice and consumed for healing purposes (Eini, 1983, p. 698; Rouzi, 2004, p. 140). The “seven salams” is mentioned in some of the Nowruz treatises written in the Safavid period. For example, in a treatise written by Mohammad Taghi Ben Mohammad Reza Razi published in 1091 of the lunar calendar the following ritual is detailed: “As for writing of seven verses beginning with Salam, at the time of new year, these mentioned verses were written with musk and saffron on a porcelain bowl, and then rinse it with rosewater or water; everyone who drinks from it will be protected against diseases and suffering in that year.” The author then writes the name of the verses (Esmaeili, 2012, pp. 50, 51). This ritual is also mentioned in other treatises written to describe Nowruz (Esmaeili, 2012, pp. 92, 103). In some Nowruz treatises, it is recommended to write other prayers with saffron. In Sirjan, Kerman province, it is also customary to write the surah Yasin with rosewater and saffron on a porcelain bowl at the new year (Bakhtiari, 1999, p. 304). In Herat, Afghanistan, people also write prayers with saffron on paper and then drop them into a container filled with saffron water and wait for the words to dissolve. Then they drink the water for healing purposes. The tradition is called “saffron water” (Mokari, 2000, p. 220). People have also turned to amulet and charm writers to bless their gardens or ward off plant pests. Some of these prayers were also written with saffron (Saedlou and Saghaminejad, 2004, pp. 80
88; Tabatabei-Ardakani, 2001, pp. 425
427). Writing Qur’anic verses and praying with saffron on shroud is also common in some areas. These shrouds are usually taken to sacred places and tawaf performed on them (see Abrishami, 2004, p. 362).

1.3.3 Other uses of saffron Other uses of saffron, especially in rituals and beliefs, include the following: G

G G

G

G

G

G

With musk and saffron the word “dear” is written on the forehead of the bride so that her love grows in the heart of her husband (Masse´, n.d., vol. 1, p. 82). The body of a newly deceased is made fragrant with musk and saffron (see Safa, 1977, p. 30). People in Abyaneh, Isfahan province, believe that if a person is on his deathbed, to make it easier for his soul to leave his body, he must be given nutritious food. This food is dissolved in saffron water and given to the dying person to drink (Nazari-Dashli Boroun, 2005, p. 566). One of the common traditions in Lapui, a city in Fars province, is welcoming the new year, by the shepherds taking the sheep to water springs to wash them; after that they let the wool dry so that they could paint them according to their favorite; the paint was a mixture of henna, saffron, and some other natural substances (Jafari and Jafari, 2007, vol. 2, p. 49). In the port of Kang, Hormozgan province, a kind of divination ceremony is held on the fourteenth day of the lunar month. The “14th” is a women’s ceremony in which a group of women get together in a house and soak some henna into rosewater in a large porcelain bowl. Then they add some wild rue, saffron, and black currant for consecration to the bowl. After that, the women put their rings in henna. Then they ask a 4- or 5-year-old boy to mix henna thoroughly. Meanwhile, they start to sing poems. The child takes out the rings one by one. The poem which is sung while the boy takes out the ring is considered to be the fortune of the owner (Daryaei, 2004, p. 69). According to the people of Nesa, Fars province, a piece of charcoal should be placed next to the container of saffron for the jinns not to take saffron away (Rezaei, 2008, p. 459). In some areas, people light candles, musk, and saffron at the bed of a sick person, then they pat him in the back and say “may your pain and calamity go to the desert, go to the sea” (Hedayat, 2001, p. 54).

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Saffron is also mentioned in literary and historical texts: G

G

In the text Shahnameh, burning of saffron is mentioned along with other fragrant substances during celebrations and even mourning (see Ferdowsi, 2007, vol. 1, p. 89; vol. 4, p. 308; vol. 5, p. 459; vol. 6, p. 595; vol. 7, p. 274). These examples are also found in epopees such as Kushnameh (see Matini, 2005, pp. 279, 455, 579). In other Persian and Arabic texts, there are many examples of saffron applications (see Abrishami, 2004, pp. 374
384). A makeup made of saffron was commonly worn by the emirs and sultans (see Bahar, 2007, pp. 286, 297).

1.3.4 Popular medicine Popular medicine or folk medicine is a part of folklore, customs, and rituals. Of course, popular medicine is different from traditional medicine (for more information on this, see Janebollahi, 2011, pp. 400, 401; Matin, 2010, pp. 61
70, and Chapter 25: Saffron in Persian traditional medicine). The difference between these two domains can be compared to the differences between classical literature and popular literature. Just like popular literature and classical literature are connected, popular medicine and traditional medicine have also been influenced by each other. Traditional medical practitioners sometimes use saffron in their treatments, but that will not be discussed here (for more information Avicena, 1988, vol. 5, pp. 145, 146; Zaryab, 1991, pp. 311
313). In public medicine, saffron has many uses. In Gilan province, saffron, rosewater, and opium are mixed together to treat earaches (Payandeh, 1977, p. 255). In Khorasan, for hard of hearing or “trapped wind in the ear,” saffron is mixed with raw oil and put in the ears (Shakurzadeh, 1984, p. 129). In Sarvestan, Fars province, in order to prevent epileptic episodes in children, sugar, saffron, and quinine is mixed together in balls the size of peppercorns and given to children three times per day (Homayouni, 1992, p. 440). This type of treatment is common among the people of Garrous (Bijar and the suburbs), Kordestan province, where children are also given rewed saffron and valerian mixed with sugar (Hashemnia and Malek-Mohammadi, 2001, p. 235). In public medicine, consumption of saffron by pregnant women is prohibited (Bazrafkan, 2010, p. 140; Saadvandian, 2010, p. 73), because there is a long-held belief that saffron induces abortion. In Tehran, during the Qajar period, if a woman opted to have an abortion for any reason, a traditional women’s doctor would prescribe her half a mithqal (4.608 g) of saffron dissolved in water per day, or a cup of barberry and saffron juice (Saadvandian, 2010, pp. 247, 316). The same method was used in Khorasan (Shakurzadeh, 1984, p. 92). In Lorestan, a mixture of saffron and honey was used (Asadian-Khorramabadi et al., 1979, p. 252). In traditional medicine, it was also believed that saffron was useful in resolving prolonged labor, as Ghazvini wrote in his book, Ajayeb al-Makhlouqat: “If saffron is administered to a pregnant woman, she will immediately give birth” and adds, “If delivery becomes problematic for a woman, they feed her saffron and she will quickly deliver her baby, which is a strange property” (Saboohi, n.d., p. 261; see also Momen-Tonkaboni, 2011, p. 448; Taghi-Mir, n.d., p. 199). This has led to the use of saffron prescriptions as charms in the occult sciences for prolonged labor, as noted in the Safineh of Tabriz, written in 722 AH/CE 1322: “The charm for a desperate woman with prolonged labor” mentioned: “Write ‘Ya Vedud, Ya Vadud’ with saffron on a water basin, and give her a cup of water therefrom to drink, immediately her pain will go away and make it easy for her to deliver” (Tabrizi, 2002, p. 246). In Khorasan, one of the ways to remove the placenta after giving birth is to eat a confection made with saffron (Shakurzadeh, 1984, p. 99). Saffron is also used for the treatment of impotency in the following areas. As in Lorestan, egg yolk is cooked with saffron and given to people (Asadian-Khorramabadi et al., 1979, p. 274). In Sarvestan, Fars province, saffron is mixed with starchy halvah and eaten (Homayouni, 1992, p. 336). In Kish island the amulet-writing master boils seven eggs and removes their skin and then he writes prayers on the eggs with saffron solution and the patient eats all of them once (Mokhtarpour, 2006, p. 270). In Khor and Biabanak, Isfahan province, saffron is recommended for the treatment of lower back pain (Hekmat-Yaghmaei, 1991, p. 398) and in Arsanjan, Fars province, a starch and saffron halva mixture (Rahimi and Hashemi, 2009, vol. 1, p. 551) is prescribed. In Minab, Hormozgan province, saffron is used to treat oral infections in children (Saeedi, 2007, p. 413). In Tehran of the Qajar period, people ate barberry with saffron as jam for the treatment of “liver swelling” (Shahri, 1999, vol. 6, p. 119). Saffron medicine is also used to aid new mothers. For example, in Khorasan, immediately after the placenta is removed, a Kachi bowl with cumin, cardamom, and saffron added is fed the new mother (Shakurzadeh, 1984, p. 99). In Tehran, ghavoot foufel was considered absolutely necessary to be given to the mother and saffron is one of the main ingredients of this dish (Saadvandian, 2010, p. 88). In Tehran, ghavoot foufel on the sixth night after the baby was born is given to the new mother. Also, a type of kachi was given to new mothers after post delivery shower to recover their power (Katiraei, 1969, pp. 79, 80, 86). Some of these foods still are used in this way particularly in remote areas.

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In Lorestan, a mixture of warm-tempered herbs including saffron in oil was fed to new mothers (Shadabi, 2016, p. 86). Saffron was also believed to be useful for melancholia treatment, as noted in historical texts (Sotoudeh, 1989, pp. 112, 113; Brown, 2003, pp. 140, 141; Haj-Javadi, 1993, p. 202). Saffron is also used to prevent miscarraiage. In Khorasan, if a pregnant woman bled for any reason, a little saffron and a few drops of water were mixed and then applied around the woman’s waist and on the navel (Shakurzadeh, 1984, p. 127). In addition to the above, saffron is used in ceremonies held for the treatment of ahl-e hawa (possessed persons) in the islands and ports of southern Iran. Before this ceremony, the treating person who is called a babazar or mamazar (depending on his/her sex) rubs a variety of medicines, one of which is saffron, on the patients skin (Sa’edi, 1977, p. 52).

1.4

Saffron in popular literature

Saffron is mentioned throughout popular literature, including in do-bayti (a two-couplet poem), riddles, fables, and stories, as discussed in the following.

1.4.1 Do-bayti In some oral do-bayti, poets use saffron to describe their despair: My sturdy body is now bent due to the separation, and my heart has now become so weak because of separation/My ruddy face that you remember, is now saffron yellow as I am separated from you. Mihandoust (2001, p. 38); for other narratives of this do-bayti; see, p. 109, Naseh (1994, p. 53). Separation has made me look yellow, like saffron, I am covered in blood like a red rose for I am away/My heart is now torn like hundred petals of flower; I have fallen to the ground like a violet flower as I am away. Mihandoust (2001, p. 72).

Saffron is also mentioned in quibbles, narrated differently in different regions of Iran: I was not in love, but you made me fall in love, I was musk and you made me saffron/I was musk accompanied by saffron, you made me lose face to the friends and enemies. Jahani-Barzoki (2006, p. 175), Naseh (2000, p. 236).

In another do-bayti recorded in Fars, the poet describes his beloved this way: I am grass and my beloved wears green, I am flower picker and my beloved is a florist/Her eyes are weighting platforms and her eyebrows the scale and she sells flowers at the price of saffron. Faghiri (1963, p. 44).

Likening expensive things to saffron can be seen in mashkzani (making dairy products by shaking milk in a waterskin) songs as well. Rural and nomadic women sing songs during mashkzani (making dough and separating butter). One of the poems sung by the nomadic women around Garmsar, Semnan province, is as follows: O dear waterskin, keep moving, become blessed by God and give four mounds of butter/Butter was expensive last year, as expensive as saffron. Shah-Hosseini (2006, pp. 228, 229).

In cuddling and lullaby songs mothers sing to their children saffron is mentioned in this way: Why didn’t you plant cotton, cotton is expensive, as expensive as saffron/A long garment for boys, and one scarf (chador) for girls. Hekmat-Yaghmaei (1991, p. 416).

Saffron is also reflected in wedding songs sung by women at various wedding stages such as henna night, marriage conclusion, and the ritual of following the bride and bridegroom to their house (arooskeshan). For example, in Neiriz, Fars province, when going to the bride’s house to bring her to the bridegroom’s house, people sing poems such as the following: I crushed cumin and let it dry, the expensive cumin of Kerman/New we are off bringing a woman, a woman at the dear price of saffron. Homayouni (2000, p. 116).

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In Sirjan, Kerman province, when setting up the consummation bed, the bridegroom’s sister sings: I will plant flowers before the bed and saffron behind the bed/ Sometimes I will visit for the smell of flower, sometimes for the smell of saffron Moayyed-Mohseni (2002, p. 118).

1.4.2 Riddles Saffron is also the subject of some of the riddles of popular literature, such as those recorded in Kouhmareh, Fars: It is yellow, not apricot/fleshy, but not peach/it is in the garden of a certain Khan/is the seasoning of the elders. Behrouzi (2001, pp. 159, 160). A narrative of this riddle is also found slightly different in Khorasan Shakurzadeh (1984, p. 539).

Or this riddle that is common in different parts of Iran: It is yellow or yellowish, bulbul makes a felt hat/It is grown in Khorasan, but eaten by the elders. Behrouzi (2001), Emam-Dezfouli (2000, p. 101), Hashemi (2011, p. 46), Homayouni (2000, p. 383); for more familiarity with the more riddles on the theme of saffron. Behrouzi (2001, pp. 159, 160), Emam-Dezfouli (2000, pp. 100, 101).

1.4.3 Stories and humor In a humorous manifest, a man does not want to accept that his wife has a lover. An old man bets on a hundred mounds of saffron to prove this. The old man wears his dervish attire, and takes him to his house on his shoulder while the husband is wrapped in felt. The man sees his wife with her lover. The old man, shaking the felt on his shoulder, says: “Look, look at the wickedness of women/little donkey! Give me my hundred mounds of saffron” (Marzolph, 1992, p.208). The satirical story of saffron in Sanaei’s Hadiqat-al-haqiqah has also been said to be completely rooted in folklore. In this story, someone asks an ignorant person if he has seen saffron or heard something about it. The ignorant person replies: “So far, I’ve eaten it more than a hundred times with yogurt.” That person says: It’s clear that you do not even know onion, then why would you keep moving your mouth in vain? Modarres-Razavi (1995, p. 71).

This joke is mentioned in Baharestan (Hakemi-Vala, 2001, p. 96) and Latayef-al-tavayef (Golchin-Maani, 1967, p. 143), and an oral narration was recorded in Khormoj Bushehr (Jafari-Ghanavati, 2014, pp. 155, 156). Saffron is also reflected in a large number of proverbs as in the following examples: G

G

G G G G G G

G G G

G G

Not every donkey deserves saffron: A bad-natured person does not deserve a good position, wealth property, and kindness (Bahmanyari, 1990, p. 482; Dehkhoda, 1985, vol. 3, p. 1361; Zolfaghari, 2011, p. 753). What does the donkey know about the price of saffron? (Bahmanyari, 1990, p. 438; Dehkhoda, 1985, vol. 3, p. 1170). It is all the same to a donkey whether you load it with saffron or hay. Even without eating saffron his lips and mouth are yellow. Crying is better than the laughing that is caused by saffron (Shakurzadeh, 1984, p. 731). I will throw flowers for those who come, and saffron for those who do not. Carrying saffron to Ghaen, is like carrying cumin to Kerman (or like carrying coal to New Castle). A lot of saffron gets eaten without care (meaning when there is too much of something, it loses its value; Zangooei and Naseh, 2015). I am so unfortunate, as I have planted saffron but grown dung (Bahmanyari, 1990, p. 81). He has eaten rice with fish skin, but burps as if he has eaten saffron (Zolfaghari, 2009, p. 642). He eats saffron in such a way that his lips do not become yellow. It means he is a master in what he does (Zolfaghari, 2009, p. 784). Donkey does not care about the smell of saffron (Zolfaghari, 2009, p. 883). Dogs do not deserve animal oil, donkeys do not deserve saffron (Zolfaghari, 2009, p. 1100).

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There are many other examples in various texts. It is noteworthy that the frequency of saffron-related parables in the south of Khorasan particularly in Ghaen, is much higher than in other parts of Iran. (see also Mokhtari et al., 2012, pp. 129, 130; see also Zangooei and Naseh, 2015, vol. 2, pp. 874, 1175). It is quite understandable as Ghaen has been the main center for saffron production in Iran for centuries.

1.5

Conclusion

Saffron has a great influence on the culture of people living in saffron-growing areas. This effect on the popular culture is both related to the planting, growing, and harvesting of saffron and also to food, prayer, popular medicine, literature, etc. One of the most important fields of research about saffron is the comparison of saffron and popular culture in different countries.

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1649. Daniels, C.L., Stevans, C. (Eds.), 2003. Encyclopedia of Superstitions, Folklore, and the Occult Sciences of the World, Vol. 2. University Press of the Pacific, Honolulu, HI. Daryabandari, N., 2006. Cooking Book. Karnameh Press, Tehran (in Persian). Daryaei, A., 2004. Popular Rituals and Folklore of Bandar-e Kang. Ehsan Publications, Tehran (in Persian). Dehkhoda, A.A., 1985. Proverbs and Verdicts. Amirkabir Publication, Tehran (in Persian). Eini, S., 1983. Notes. Agah Press, Tehran (in Persian). Emam-Dezfouli, M.A., 2000. Dezfouli Riddles. Office of Cultural Research Press, Tehran (in Persian). Esmaeili, E. (Ed.), 2012. Twelve Treatises on Nowruz (Nowruznamah). Farsi-Tajik Folklore Research Institute, Dushanbe (in Persia). Faghih-Mohammadi Jalali, M.M., 2003. Appearance of Quchan. Bakhshayesh Publications, Qom (in Persian). Faghiri, A., 1963. Local Songs. Mohammadi Publications, Shiraz (in Persian). Fassaei, R. (Ed.), 2003. Koliyate Boshagh Atameh shirazi. Fars Studies Press, Shiraz (in Persian). Ferdowsi, A., 2007. Shahnameh. Center of the Great Islamic Encyclopedia Publications, Tehran (in Persian). Ghaem Magham Farahani, S.A., 1987. Eqamah al-Borhan Ala Osoul-e Din-e Islam. Vahid Publications, Tehran (in Persian). Golchin-Maani, A. (Ed.), 1967. Latayef al-Tavayef. Eqbal Publications, Tehran (in Persian). Haj-Javadi, S.K. (Ed.), 1993. Zobdah al-tavarikh. Ministry of Culture and Islamic Guidance Press, Tehran (in Persian). Hakemi-Vala, E. (Ed.), 2001. Baharestan. Ettelaat Publication, Tehran (in Persian). Halvorson, S., 2008. Saffron cultivation and culture in central Spain. Focus Geogr. 51 (1), 17
24. Hashemi, A., 2011. Riddles, Accepted by Yesterday, Neglected by Today. Soroush Press, Tehran (in Persian). Hashemnia, S.M., Malek-Mohammadi, M., 2001. Culture of Garrous. Moallefin Press, Tehran (in Persian). Hasouri, A. (Ed.), 2005. Nowruznamah. Cheshmeh Publications, Tehran (in Persian). Hatami H., Beliefs and Behaviors/Previous in Kazeroun, 2006, Tehran, (in Persian). Hedayat, S., 2001. Iranian Popular Culture. Cheshmeh Publications, Tehran (in Persian). Hekmat-Yaghmaei, A., 1991. At the Coast of Salt Desert. Toos Publication, Tehran (in Persian). Hekmat-Yaghmaei, A.K., 2013. Economic life and livelihood of the people of Khor and Biabanak in the late Qajar period. Payam Baharestan 1 (in Persian).

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Homayouni, S., 1992. Culture of Sarvestan. Astan-e Quds Razavi Publications, Mashhad (in Persian). Homayouni, S., 2000. Fars Local Songs. Foundation for Fars Studies Press, Shiraz (in Persian). Hutchings, J., 2004. Color in folklore and tradition—the principles. Color Res. Appl. 29 (1), 57
66. Izadi, M.R., Ranjbar, H., Amini, A.Gh, 1994. The Land and Culture of the People of Izadkhast. Ayat Press, Ahvaz (in Persian). Jafari, B., Jafari, R., 2007. Lapui, Brilliant Star of Fars. Ilaf Publications, Shiraz (in Persian). Jafari-Ghanavati, M., 2014. Sugar Bowl and Salt Shaker. Ghatreh Press, Tehran (in Persian). Jahani-Barzoki, Z., 2006. Barzok, Gem of the Mountain. Maranjab Publications, Kashan (in Persian). Janebollahi, S., 2011. Popular and Traditional Medicine of Iranian People. Amirkabir Publication, Tehran (in Persian). Kakisis, J.D., 2017. Saffron: from Greek mythology to contemporary anti-atherosclerotic medicine. Atherosclerosis 268, 193
195. Kashefi-Sabzevari, M.H., n.d. Asrar-e Ghasemi. Pishawar Publications, Tehran (in Persian). Katiraei, M., 1969. From Clay to Clay. Institute for Social Studies and Research, Tehran (in Persian). Khaleghi-Motlagh, J. (Ed.), 2007. Shahnameh. Center of the Great Islamic Encyclopedia Publications, Tehran (in Persian). Khavar, Z., 2009. Cooking Art in Gilan. Ataee Press, Tehran (in Persian). Magdalini, A., 2017. Development of a Business Plan for a Social Cooperative Enterprise (SCE) with Healing Organic Plant Products and Herbs through an Online Venture (MSc. thesis). International Hellenic University, Thessaloniki. Marzolph U., Typology of Persian Folk Tales, (Jahandari K. Trans.), 1992, Soroush Publication, Tehran, (in Persian). Masse´, H., n.d. Iranian Customs and Beliefs, (Roshanzamir, M. Trans.). Shafiei Press, Tabriz (in Persian). Matin, P., 2010. Anthropology in Medicine. Safir-e Ardehal Publications, Tehran (in Persian). Matini, J. (Ed.), 2005. Koushnameh. Elm Publications, Tehran (in Persian). Mihandoust, M., 2001. Kaleh Faryad. Golazin Publications, Tehran (in Persian). Moayyed-Mohseni, M., 2002. Sirjan Popular Culture. Kerman Studies Center, Kerman (in Persian). Modarres-Razavi, M.T. (Ed.), 1995. Hadiqat al-Haqiqah. University of Tehran Press, Tehran (in Persian). Mokari, M., 2000. Nowruz in Herat, Afghanistan (Collected Articles of the First Nowrouz Conference). Anthropology Research Institute, Tehran (in Persian). Mokhtari, A., Meghdari, S.S., Mokhtari, H., 2012. Saffron in Mithqal. Akbarzadeh Publications, Ghaen (in Persian). Mokhtarpour, R., 2006. In the Year with Kish Natives. Varjavand Publications, Tehran (in Persian). Momen-Tonkaboni, M., 2011. Tohfe-al-Momenin. Natural Resuscitation Institute, Qom (in Persian). Moshiri, M. (Ed.), Ershad Al-Zeraah, 1967, Amirkabir Publication, Tehran (in Persian). Mostowfi, A., 1992. A Description of My Life. Zavvar Publications, Tehran (in Persian). Naseh, M.M., 1994. Beloved Poetry (Birjand Pupular Do-bayti). Mohaghegh Publications, Mashhad (in Persian). Naseh, M.M., 2000. Poetry of Sadness (Birjand Popular Quatrains). Astan-e Quds Razavi Publications, Mashhad (in Persian). Nazari-Dashli Boroun, Z., 2005. Anthropology of Abyaneh Village. Anthropology Research Institute, Tehran (in Persian). Nezami-Ganjavi, 1995. Koliat-e Khamseh. Amirkabir Publication, Tehran (in Persian). Payandeh, M., 1977. Rituals and Beliefs of Gil and Deilam. Iranian Culture Foundation Press, Tehran (in Persian). Rahimi, H., Hashemi, S., 2009. Buoy of Arsanjan. Asar Daneshvaran Publications, Qom (in Persian). Rezaei, G., 2008. My City Fasa from Another View. Navid Shiraz Publications, Shiraz (in Persian). Rouzi, A., 2004. Nowruz in Vararavdan. Anthropology Research Institute, Tehran (in Persian). Saadvandian, S. (Ed.), 2010. Memoirs of Munes al-Dowlah. Zarrin Publications, Tehran (in Persian). Saberi-Eftekhari, A., 2014. “Charm (T’aviz)”, Encyclopedia of Iranian Culture. Center of the Great Islamic Encyclopedia Publications, Tehran (in Persian). Saboohi, N. (Ed.), n.d. Ajayeb al-Makhlouqat. Naser Khosrow Publications, Tehran (in Persian). Saedlou, H., Saghaminejad, M. (Eds.), 2004. Mafatih al-Arzaq or Keys to the Gohar Treasures. Cultural Heritage Society, Tehran (in Persian). Sa’edi, G.H., Ahl-e Hawa, 1977, Amirkabir Publication, Tehran, (in Persian) Saeedi, S., 2007. Popular Culture of Minab. Aelshan Publications, Tehran (in Persian). Safa, Z. (Ed.), 1977. Darabnamah. Book Publishing and Translation Agency, Tehran (in Persian). Sarmad, G., 2003. Once Upon a Time, Local Birjand Tales. Pezhvak Farhang Publications, Tehran (in Persian). Shadabi, S., 2016. Popular Culture of Lorestan. Shapourkhast Publications, Khorramabad (in Persian). Shafiee-Kadkani, M.R. (Ed.), 2005. Asrar al-Tawhid. Agah Press, Tehran (in Persian). Shah-Hosseini, A., 2006. Khowar (Garmsar) and Its Ancient Heritage. Bostan Andisheh Publications, Semnan (in Persian). Shahri, J., 1999. Social History of Tehran in the 13th Century. Rasa Cultural Service Institute, Tehran (in Persian). Shahri, J., 2004. Tehran-e Qadim [Old Tehran]. Moein Press, Tehran (in Persian). Shakurzadeh, E., 1984. Beliefs and Customs of the People of Khorasan. Soroush Press, Tehran (in Persian). Shariatzadeh, A.A., 1992. Culture of Shahrood. Shariatzadeh, Tehran (in Persian). Sotoudeh, M. (Ed.), 1989. Asar wa Ehya. University of Tehran Press, Tehran (in Persian). Srivastava, T.N., Rajasekharan, S., Badola, D.P., Shah, D.C., 1985. Important medicinal plants of Jammu and Kashmir I. Kesar (Saffron). Anc. Sci. Life 5 (1), 68
73. Tabatabei-Ardakani, S.M., 2001. Popular Culture of Ardakan. Ministry of Culture and Islamic Guidance Press, Tehran (in Persian). Tabrizi, A., 2002. Safine-ye Tabriz. University of Tehran Press, Tehran (in Persian).

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Taghi-Mir, M. (Ed.), n.d. Ekhtiarate Badiei. Razi Distribution Company, Tehran (in Persian). Unwala, R., 1922. Narrations Darbnameh of Hormozdiar. Bombay (in Persian). Vakilian, S.A., 2001. Ramadan in Iranian Culture. Soroush Press, Tehran (in Persian). Zangooei, H., Naseh, M.M., 2015. Proverbs and Verdicts of the People of Southern Khorasan. Fekr-e Bekr Publications, Birjand (in Persian). Zaryab, A. (Ed.), 1991. Al-Saydanah. Iran University Press, Tehran. Zolfaghari, H., 2009. Great Encyclopedia of Persian Proverbs. Moein Publications, Tehran (in Persian). Zolfaghari, H. (Ed.), 2011. Jame al-Tamthil. Moein Publications, Tehran (in Persian).

Further reading Aristeridou, M., 2017. Development of a Business Plan for a Social Cooperative Enterprise (SCE) with Healing Organic Plant Products and Herbs through an Online Venture (MSc. thesis), International Hellenic University, Thessaloniki., Homayouni, S., 2004. Old Tehran. Moein Publications, Tehran (in Persian). Jafari-Ghanavati, M., 2016. Khezr. Encyclopedia of Iranian Culture, Tehran (in Persian).

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Chapter 2

Saffron and religion Mansour Motamedi1, Fayyaz Gharaei1 and Salman Saket2 1

Department of Religion and Comparative Mysticism, Faculty of Theology and Islamic Studies, Ferdowsi University of Mashhad, Mashhad, Iran,

2

Department of Persian Language and Literature, Faculty of Letters and Humanities, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 2.1 Introduction 2.2 Saffron in Indian religions 2.2.1 Saffron symbols 2.2.2 Saffron in Tantara rites 2.2.3 Saffron for laundering Gods 2.2.4 Saffron color in India’s flag 2.2.5 Saffron, the color of religious costumes 2.2.6 Saffron celebrations and festivals

2.1

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2.2.7 Saffron color in Buddhism 2.3 Semitic religions 2.3.1 Judaism 2.3.2 Islam 2.4 Conclusion References Further reading

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Introduction

Studying the role and status of an agricultural product in any religion may at first seem strange. However, having recalled the fact that religion, as an important dimension of culture, is related to all aspects of individual and collective life and plays a role in all daily life shows it is easy to see how saffron has deviated from its mere agricultural function and has acquired a bold cultural hue in the history of some regions. In the present chapter, considering the subject and geographical region, we focus on three groups of religions. The first group is the Persian religions, which consist of Zoroastrian, Manichaean, and Mazdakite. However, due to lack of evidence related to the role and status of saffron in Manichaeism and Mazdakism, inevitably they are not discussed. In Zoroastrianism, saffron is discussed based on Avesta references and other Zoroastrian texts. Due to the widespread use of saffron in various aspects of the culture of Iran, which may well reflect the long history of the role of this aromatic plant in Iranian social and religious life, the use of saffron in the Zoroastrian religion is discussed in the section “Saffron in pre-Islamic Iran” in Chapter 3, Saffron in the Ancient history of Iran, of this book. The second group is the Indian religions, which are comprised of Hinduism, Buddhism, and Jainism. The third group is the Semitic religions, the religions of Abraham, which include Judaism, Christianity, and Islam. Among these three religions, saffron is solely mentioned in the religious texts of Judaism, and has appeared in both the original sacred text of Judaism, the Old Testament, and also in its expositions, especially in Talmudic texts. However, saffron is not mentioned in the original sacred texts of Christianity and Islam. Indeed, the Jews, whether at the time of writing and compiling the Old Testament or in the later periods, had an especial connection to the East particularly to Iran. The later flourishing of Christianity occurred in places where saffron was not popular, which is probably why it does not in the sacred texts and culture of the religion. While there is no mention of saffron in the Islamic text the Quran there are many references to it in the hadiths (Islamic traditions), indicating its prevalence in Iranian society and culture. Despite the fact that this sudden and abundant attention given to saffron in hadith texts helps the researcher become familiar with the various angles of cultural, social, and religious uses of saffron, it breeds concern that since it is away from the beginning of Islam in terms of time and from its origin in terms of place, the probability of

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00002-2 © 2020 Elsevier Inc. All rights reserved.

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misrepresentation and forgery of later hadiths increases. For this reason, here we only briefly mention the hadiths. Since the review and analysis of this process, as well as distinguishing the valid hadiths from the invalid ones are not discussed in this study, there has been only a brief mention of these hadiths.

2.2

Saffron in Indian religions

In the Sanskrit language, saffron is called kesaravara (केसरवर) and in the Hindi language it is called kesar () from the Sanskrit root word kesara (), which means hair and eyebrow, and refers to thin strands of saffron. Another Sanskrit word for saffron is ka´sm¯ırajanman (कश्मीरजन्मन्). The suffix janman () means bearing and origin, so ka´sm¯ırajanman means born in Kashmir or Kashmiri origin, which is sufficient proof that Kashmir was believed to be the birthplace of saffron. Sometimes saffron is referred to as crocus (फूल) in Hindi, which of course means ornamental saffron, a plant from the Iridaceae family. Saffron has applications in India and all Indian religions (Buddhism, Jainism, Sikhism, and especially Hinduism), in their temples (from Mandir to Gurdwara), religious costumes, rituals, medicine and therapies, cooking and preparing of food, and clothes. Saffron is also mentioned in prominent Hindu books such as Mahabharata and Ramayan. In Indian medical texts on traditional medicine called the Ayurveda saffron is used as an antidepressant and to treat muscle cramps (spasm), bronchitis, sore throats, headaches, fever, and to raise the body temperature of pregnant women as well as a sexual stimulant (Swami, 2005, p. 111). Because of its stimulating effect, it was used by grooms at the time of consummation. It is a popular alternative medicine and has a thriving lucrative market in India. Of course, saffron was also used to dye important garments. Of course, saffron was also used to dye important garments. However, these are the prevalent uses of saffron and are more or less common in most cultures, and what is more important is its application in rituals and rites

2.2.1 Saffron symbols One of the most important ritual applications of saffron is to paint the forehead and hair part of women as a religious symbol. If this saffron dot is on the forehead, it is called t¯ılaka (तिलक) in Sanskrit and t¯ık¯a (टीका) in Hindi, and is an indication of religious, sectarian, and spiritual affiliations. If this symbol is on the hairline and part, it is called sind¯ura () in Sanskrit and sind¯ura (सिन्दूर) in Hindi, which means red color and is a sign of marriage. The other sign appears between the two eyebrows and is called bindu (बिंदु) in Sanskrit and bindi (बिंदी) in Hindi and means dot or mole, referring to the third eye of Shiva (one of the triple Indian Gods who is the manifestation of God’s glory and invincibility and the God of mort), which he opens at the time of the destruction of this world drowned in darkness for a fresh start. This mole is a sign of insight, which is mostly regarded as a symbol of women’s beauty. These moles and signs are as old as Hinduism and other Indian religions. In ancient historical and literary Persian works, they are referred to in various ways, such as mole and ghashghah (1. a dark spot a horse’s forehead 2. A sign put on the forehead) and their applications in religious rituals are noted (Dehkhoda, 1963). In addition, Indian and Iranian Persian poets have used these signs to describe themes. In the 10th CE, to describe the saffron signs of Hindu clergymen (Brahmans), Masoudi says: “Male and female Brahmans hang yellow strings like sword belt on their necks to be distinguished from other Hindus” (Masoudi,1968, vol. 1, p. 71). Unsuri Balkhi (CE 9th and 10th) in the mention of a saffron ornament worn by Indians says: No one grows saffron in India/ since its dignity is to be used as ornament. Because your solemnity gives dignity/ every year without saffron breeds a pale look. Onsori Balkhi (1984, p. 43)

Describing a good-looking Hindu boy at the time of Timurid dynasty, when the ritual of rubbing saffron on the body was common, Ashraf Mazandarani (17th CE) says: Similar to Iris, abir is sprayed from bosom to navel/resembling narcissus, pleated skirt full of saffron to waist. Ashraf Mazandarani (1994, p. 265)

In CE 16th in Jahangir, the Indian Gurkani King, while visiting Koubandval in India, noted that: “There was a Hindu named Erjen dressed up as an old virtuous sheikh who rubbed a pinch of saffron, which is called ghashghah and believed to be auspicious by Indians, on a king’s forehead” (Jahangir Gurkanid, 1980, p. 42).

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In some travelogues of people who had traveled to Isfahan (in Iran) and the area of Oulala in India in CE 17th and also in Persian poems of that century and the following centuries there were mentions of this role of saffron (Abrishami, 2002, p. 341). One of the most famous references to this sign is in Hafiz’s poem (Hafez,1992), which says: That beautiful Shirazi Turk took control and my heart stole/I’ll give Samarkand and Bukhara, for her Hindu beautiful mole. One of its meanings is the beautiful mole, which originally belongs to the Indians, between the eyebrows. Apparently, even at the time of Hafiz when Shiraz had maritime business exchanges with India the Indian mole was used to express the beauty of women. Banyans, the Indian merchants, used saffron in their customs and traditions. For example, Pietro Della Valle in 1025 AH (CE 1617) in describing their ceremonies says: “Banyans are all dressed in white from head to foot at the time of their celebrations. However, their costumes are painted dark yellow on the chest with saffron and their turbans are dyed with the same substance. Inside the yellow color, red spots have been put using a sandal and they are so fond of these two colors that they have even put them on their foreheads using the same style”(Della Valle, 1969, p. 72).

2.2.2 Saffron in Tantara rites Applying saffron in one of the well-known Indian religious rites called Tantara and performing its rites to awaken hidden power and bodily strength (kundalini) is a necessity. Tantara symbolism is very important and mysterious. "One common element of Tantara is using sacred geometric cosmological designs named “yantra” and “mandala.” In Tantara Buddhism, which has propagated sophisticated designs, the expression of Mandala, which means circle of influence, is seen repeatedly. Yantaras are tools and devices designed to preserve and visualize divine power. Yantaras might be designs drawn on paper, etched in stone and iron, or preserved in other things. They may also be 3D visualizations. In many temples of Tantara, the “true” symbol of God is a Yantara embedded in the interior shrine that is not visible and accessible to everyone. Typically, Yantara has a central point called the “bindu.” This is the symbol of degradation of the high world in the circle of manifestation. . . .Sometimes B¯ıja Mintara whose Yantara is God, may appear under Bindu. . . .The upward triangle in this symbol is a sign of masculinity (linga) and the downward triangle implies femininity (yoni). . . . The well-known Yantara consists of five downward and four upward intersecting triangles and is essentially viewed as the symbol for unity of feminine and masculine essence. The same as a drop of water, which causes an outward ripple when it drops on the surface of a pond, the petals can also be seen as the sparkle of creation and flourishment of germinated seeds (B¯ıja). Therefore Yantara is considered the position of God" (Rodriguez, 2006, pp. 266
267). Saffron is commonly used to paint these important religious symbols.

2.2.3 Saffron for laundering Gods Another application of saffron is for laundering the idols. As the conquest of Sumanat and breaking of the idol “Manat” by Mahmud Ghaznavi has been described in Persian sources, this Hindu tradition is referred to as well (Abrishami, 2002, p. 343). In Hinduism, the renowned idols of Shiva, Barhama, Vishnu, and female goddesses like Kali and Mahadiu are decorated with flowers and smeared with saffron and aromatized with fragrance and musk in various ceremonies, particularly Shaktis (a sect of Shiva worshippers) (Kay Khosrow Esfandiar, 1983, vol. 1, p. 167). Smearing the Hindu God’s idols with saffron colored the faces and heads of the followers. As Mohammad Fadaei (CE 18th) in the verse story of “Khoram and Ziba” while referring to ceremonies in idol houses says: “. . .he smeared saffron on the forehead and ear/ like a Hindu coming from the idol house”(Fadaei, 2015, p. 79). On rubbing saffron on the groom’s body at the wedding, the Iranian Aqa Mohammad Behbahani during his journey in India between 1902 and 1907 wrote: “On Indian wedding. . . at the time of wedding, a few days before henna ceremony, saffron and turmeric are rubbed on the bride and groom’s bodies and they are dressed in yellow costume and then are seated in a secluded place, during this time they do not leave the place very much” (Kermanshahi, 1993, p. 111).

2.2.4 Saffron color in India’s flag India’s flag consists of three colors, which are similar to Iran’s flag with a difference in the arrangement of colors, which are in reverse order, and saffron color is used instead of red. In India, which is dominated by Hindus (80%), the saffron color is one of the three colors and seemingly the symbol and representative of Hindu population, whereas the

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other two colors are green and white. In India’s flag, saffron is on top, green is at the bottom, and white is in between, which is an indication of superiority and domination. But Ali Asghar Hekmat who was the Iranian ambassador to India in 1957 on selecting the colors for India’s flag writes: “In accordance with the constitution, India’s flag is comprised of three colors, saffron, white and green arranged horizontally from top to bottom, merged equally. . . there is an entertaining anecdote behind selecting these colors and that is, in 1921 the general committee (assembly) of the congress party chaired by Mahatma Gandhi was held, meanwhile a young man from that region conferred upon Gandhi a flag consisting of red and green symbolizing Hindu and Muslim. This appealed to Gandhi and he added a white part to the flag to be the representative of other Indian religions, also he added the image of wheel, which is the secret of movement and progress. Later, in 1931, when the general committee of the congress party agreed on these three colors to be the national flag, it was stated that the colors did not have anything to do with religious titles, but that saffron is the sign of bravery, white is the sign of honesty and peace, and green is the sign of faith" (Hekmat, 1958, p. 403).

2.2.5 Saffron, the color of religious costumes The color saffron and its derivatives is the most common religious and ritual color in India. The costumes of students, disciples, fakirs, religious, and sacred figures and even political personalities are saffron. The flags of religious groups are also the same color. This color seems to be widely accepted by the general public and is even used by religious and Islamic political figures as well as in ethnic rituals in India. Some Muslim mystics who traveled to India and resided there wore yellowish saffron attire. Lali Badakhshi in a biography about Nour Al-din Malek Yar Parran, a settler in Delhi (died in CE 1281), noted that: “He is originally from Lar and a follower of Sheikh Danial Khalaji . . . at the time of King Ghiyas Al-din Balban he came to Delhi. . . . He used to wear yellow clothes at all times” (Lali Badakhshi, 1990, p. 1023). Moreover, some local Indian kings, following older customs, drew signs and drawings on their bodies and clothes with saffron. This tradition was common until CE 18th, and even the beautiful yellow color was considered a royal sign (Abrishami, 2002, p. 347). Muslim rulers, influenced by the dominant Hindu culture, wore saffron attire. Apparently, wearing a scarf dyed in saffron was common and it was reported that: “While Jahangir, the Timurid King of India, was wearing a saffron scarf around his neck, his Iranian wife Noor Jahan Beigom (Ghias Beig Tehrani’s daughter) told him at his sight: “there is no saffron color on your bosom/ my face’s yellow color has affected you”(Abrishami, 2002, p. 347).

2.2.6 Saffron celebrations and festivals India has two major public holidays one of which is called “Houli” and is held at the beginning of spring and the other one called Diwali is held in the fall. These two celebrations are the biggest and most important festivals of India and Hindus. Houli is the celebration of colors, and is almost coincident with the Iranian celebration of Nowruz and both have a single root. In the Houli celebration a variety of colors are used and people participating in it become rapt in dance and rejoice, temporarily discarding class distinctions and splashing paint at each other. Although different types of colors are used in these celebrations, saffron color and its derivatives are used the most. Throwing saffron during Iranian celebrations and Islamic festivals (Eids) in imitation of the Houli festival became widely prevalent at the time of Timurid, the Muslim rulers of India. In the 17th century Ashraf Mazandarani observed the Houli festival a (Abrishami, 2002, p. 382; Ashraf Mazandarani, 1994, pp. 152
153): Hey butler, unveil the hijab/ and unmask the beauty of modesty. So that I’ll narrate the story of the Indian Holy/ and begin to explain about Indian Bolfozouli. I entered the assembly like a Holy participant/ listen to me, I am reporting the event. Once again the outburst of India’s lust/ the tradition of frenzy manifested itself. Occasionally, they paint each other’s clothes by throwing red and saffron colors.

Other poets have written about the Indian tradition of saffron throwing at celebrations and eids. Monir Lahoori (died in CE 1644) (Monir Lahoori, n.d., p. 51), Gouya-ye Pinjabi Ghaznavi (died in CE 1704) (Gouya, 1963, p. 31), and Seraj Al-din Ali Arezoo (died in CE 1756) (Laleh Tik Chand, 2001, p. 1358) have written poems on the celebration of Holi and throwing saffron in it (see Abrishami, 2002, p. 383).

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As Abd Al-latif Shooshtari (18th century) writes in his Indian travelogue: “Another great festival is Houli that coincides Soltani (royal) Nowruz and lasts for a month, . . . they have a tool called twists (pichkari), whenever abir, water or something else is put and turned, the tool and whatever it contains go for several zar (a unit of measurement for length). . . I heard that there were eighteen thousand twists (pichkari) made of gold and silver used for playing” (Shooshtari, 1984, p. 379). To explain the twisting, Abrishami says: “pichkari is a compound Persian word that is currently used in Afghanistan, which means syringe and injection. Certainly the Hindi spray device was similar to needlefree syringe”(Abrishami, 2002, p. 383). As I witnessed myself (F. Gharaei, one of the authors of this chapter) in the Houli celebration in New Delhi, India in the year of 1996, small and big pumps were used to splash paint, which were sold in a variety of sizes and sometimes in sacks. In celebrations held at the time of Akbar Shah Gurkanid saffron was thrown. Sheikh Abul Fazl Mobarak Nagouri (killed in 1602 AH) in describing a celebration at Gurkanid palace held to celebrate Akbar Shah’s birthday says: “In the seraglio of infallibility and dignity arrangements have been made for exhilaration. . . beautiful nurses gave new honor to enthusiasm by throwing gulab. Smiling wearers of amethyst clothes, while throwing saffron, covered gorgeous women with gold” (Nagouri, 1993, vol. 1, p. 36). Shebli Nomani says: “One day on birthday celebration, saffron color was thrown on clothes and Sheikh Abd Alnabi (very well esteemed by Akbar and served as a guru for him) had seen and got so furious that as a result hit him with his crane”(Nomani, 1984, p. 36).

2.2.7 Saffron color in Buddhism When Buddha passed away, his corpse was wrapped in a cloak dyed in saffron and thus Buddhists also have a special interest in the saffron color. In addition, Buddhist monks’ costumes are in saffron color or its derivatives and they believe that saffron clothes take them to the desired liberation point more swiftly. According to Buddhist legend, an Indian Buddhist monk named Madhyantika was the first person who planted saffron in Kashmir (Abrishami, 2002, p. 352). Buddhists use the Mandala sign, which consists of saffron and saffron colors as the main components, more than other Indian religions.

2.3

Semitic religions

2.3.1 Judaism Throughout the entire Tanakh or the holy book of Jews, the Hebrew term karkom (‫ )כרכם‬is used in only one place (Song of Songs 14.4). The term describes a beloved friend and among numerous metaphors used about the sweetheart she is likened to an enclosed garden in which, in addition to beautiful manifestations and value-adding factors of the garden, “precious fruits” grow as well. Along with valuable crops of this garden that the lover names, he mentions “spikenard, saffron, calamus [sugarcane], cinnamon and various trees such as frankincense, myrrh and aloes and all precious spices.” Naming saffron beside sugarcane, which has a pleasant flavor and fragrances like frankincense, myrrh and incense indicate that saffron was considered as a freshener as well as a flavor. Its mention beside spikenard indicates its beauty, whether in the form of saffron flower or the beautiful color obtained from it. In addition to the holy text the Old Testament, the term karkom has appeared in the collection of “Braita” when talking about the rules of burning incense in temples. Eleven substances have been mentioned in this instruction among which there are items similar to the ones in Songs of Solomon. The common points are frankincense, myrrh, spikenard, saffron, and cinnamon (Talmud, Krithoth, 6 A, Pitum Haketoret). For a long time, based on the common tradition of understanding religious texts, Jews have considered the term karkom to be saffron. But some scholars are skeptical about this idea and have taken the word karkom to mean something else. Immanuel Lo¨w (1854
1944), a Hungarian Rabbi and a specialist in Hebrew and Aramaic etymology of plants and also translator of Songs of Solomon to Hungarian, believes that since the fragrances mentioned in both lists are crops, which are originally from far tropical regions, karkom cannot be saffron, but rather is Curcuma longa or turmeric, which grows in tropical regions and was called Indian saffron (Lo¨w, 1924, s.v.). However, this idea cannot be accepted. Because turmeric is not considered as a fragrance and is mainly a color additive and food flavor, mentioning its name beside the other aforementioned fragrances is not relevant (Feliks, 2006, s.v.). On the other hand, mentioning saffron with spikenard, aside from its aroma, implies the beauty of its flower as well. Another researcher skeptical about the implication of karkom for saffron in Songs of Solomon is Mohammad Hassan Abrishami (1939). He believes that mentioning the word saffron in Songs of Solomon is a mistake made by translators: “. . .the Yemeni saffron, which means vars‫[ ﻭﺭﺱ‬reseda], and some earlier authors have mistakenly referred

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to it as ‘Yemeni saffron’. . . in all probability, this mistake has occurred in translating ancient Greek, Hebrew and Pahlavi texts to Arabic, while the word qortom ‫[ ﻕﺭﻁﻡ‬carthamus] and Vars (and also some plants or things that had similarities to saffron in one way or another) have been translated as saffron. The present author doubts whether the word za’faran in Persian translation of Song of Songs in Torah [sic.] . . .is the same as its original Persian and Arabic meaning. Rather, the same mistake must have happened in the translation of qortom and vars (plants grown in Egypt and Yemen) as saffron or Persian karkom. Because in the territory of prophets and kings of Israelites planting the original saffron was not common, and they used the Persian word Karkom for vars, qortom and osfor ‫[ ﻉﺹﻑﺭ‬Carthamus tinctorios]”(Abrishami, 1997, pp. 21
22). Prior to mentioning this mistake, the author has provided a detailed and accurate description of the plants, which are the right replacement of saffron in Song of Songs and the reader may refer to his explanations. The main criticism of Abrishami’s point of view is that he has assumed a principle as correct if a plant is mentioned in a text, including in the Old Testament, necessarily the cultivation place of that plant must be the region where the followers of that holy book lived. On the other hand, this scholar believes that the cultivation of saffron in the regions of Levant and Palestine was done by Iranian migrants after the advent of Islam (Abrishami, 1997, p. 111) and since the text of Song of Songs belongs to centuries before the advent of Islam and migration of Iranians to those regions, there is no possibility that saffron was cultivated at that time. We do not discuss this second opinion but only say that even the Jewish researchers themselves agree with Abrishami that Iran is the birthplace of saffron (Feliks, 2006, s.v.). However, more discussion and study is required on the transfer time, but concerning the first speculation, his point of view cannot be seen as acceptable, because, as it was mentioned, referring to a plant’s name in a text does not mean that it was planted there too. In a verse from the book of Song of Songs, some fragrances like myrrh are mentioned, which were taken by merchants from southern Saudi Arabia to Canaan several centuries before the Common Era (Finkelstein and Silberman, 2015, p. 37). Moreover, we refer again to the point we mentioned in rejecting Lo¨w’s view, since the plants mentioned by Abrishami as the replacements for saffron do not have the same aroma, vividness, and beauty as saffron, and thus are not used in a romantic sonnet addressed to the sweetheart. Another term used in ancient times, which is believed by some to be related to saffron, is “ziphron” in the Book of Numbers 34.9. James Hawkes has stated the following it in the glossary of Bible: “ziphron (fragrance) is a city at the border of northern estates of Israel. . .and is probably the Za’faranah a city mid-way between Homs and Hama. . .”(Hawkes, 1998, p. 443). Abrishami in criticizing Hawkes’s idea firstly denies the meaning that he suggests and thinks it is not based on a reliable source, since as we mentioned earlier he believes that the cultivation of saffron in Syria and Palestine was done after the advent of Islam and if there is a place named Za’faranah in that region, its background cannot precede Islam. Hence, the word Ziphron does not mean “good smell” and, based on this, it cannot be used as a proof that the city of Za’faranah existed when the Old Testament was written (Abrishami, 1997, pp. 53
54). Despite that, it should be noted that Hawkes is not alone in this sense and Ziphron, in the majority of related sources, has the same meaning. (Easton, 1897, s.v.). Interestingly, in Hebrew dictionaries and lexicons, the root of the term is not observed (Hayyim, 1981), which means it is based on speculation and probably even on the etymology of Arabic language (Jones, 1990, s.v.). In the following eras, at the times of Mishnah and Talmud, the use of saffron was widespread in Palestine and Babylon. In Babylonian Talmud, in the Bava Kama treatise 81 A, which refers to the licenses needed for using pastures, springs, and roads, it is stated that people can “use plantations hedge, even farms full of saffron (Karkom),” indicating the value of a saffron farm since even walking past it and not necessarily entering it may have been allowed. In the Islamic era, from the CE 9th century, dhimm¯ıs had to have signs on their clothes to be distinguished from Muslims. This sign was called “Ghyar” in the Muslim world and it has been said that Jews, according to the Pope’s command, had to paint the sign with saffron in the Middle Ages in Europe (Lewis, 1984, pp. 25, 27; Perlman, 1965, s.v.).

2.3.2 Islam As noted earlier, Judaism, among the other three Abrahamic religions, which are Judaism, Christianity, and Islam, is the only religion that mentions saffron in its sacred text. The term karkom is not used in the Bible, which consists of the New and Old Testament, nor in the Holy Quran. But in Islam, while it is not mentioned in its holy text (Quran), it is mentioned in the hadiths. In one hadith, which is on the medicinal effects of saffron, much is said on the prohibition of smelling and eating food containing saffron during Ihram (pilgrimage). Also, there are disagreements regarding the use of clothing dyed with saffron in some texts of jurisprudence; from the point of view of some religious jurists, wearing clothing dyed with saffron is forbidden, but others believe that if the smell of saffron is removed, it is acceptable (Al-Hurr al-Aamili, 1995, vol. 12, pp. 442, 446, 449; Koleini, 1401, vol. 4, pp. 342
343 and 354
355; Moslem Ibn Hajjaj Neyshabouri, 1954, vol. 3, pp. 293
294).

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21

However, the discussion on the authenticity of hadiths requires a more specialized and extensive look, and we offer this in Chapter 3, Saffron in the Ancient history of Iran, of this book.

2.4

Conclusion

Saffron is used commonly in Hinduism. In Persian literature of the Islamic period of India, there are also frequent references to saffron indicating its wide use in India especially among the Hindus. The reference to saffron in the Old Testament shows that the Jews were familiar with it and, considering some passages of the Talmud, we can conclude that at the time the plant was being cultivated in surrounding lands. In Islam, while there is no reference in the Quran, because of the Muslims’ acquaintance with Iran’s flora, the references to saffron and its various properties found their way into the many Islamic traditions (hadiths).

References Abrishami, M.H., 1997. Iranian Saffron and a Historical, Cultural, and Agricultural Study. Astan Quds Razavi Publications, Mashhad (in Persian). Abrishami, M.H., 2002. Iranian Saffron. Amirkabir Publication, Tehran (in Persian). Al-Hurr al-Aamili, M.H., 1995. Was¯a’il al-Sh¯ıʿa. Al-Bayt Institute PBUH Lahya’ Altras, Qom (in Arabic). Ashraf Mazandarani, M.S., 1994. Divan of Ashraf Mazandarani. Literary and Historical Publishing Company of Dr Mahmoud Afshar Yazdi’s Endowments, Tehran (in Persian). Dehkhoda, A., Loghatnameh Dehkhoda, Tehran, Tehran University Press, 1342 SE./1963 CE. Della Valle, P., 1969. Pietro Della Valle’s travelogue, (Sh. Shafa, Trans.). Scientific and Cultural Publications Company, Tehran (in Persian). Easton, M.G., 1897. Eastons’ Bible Dictionary. Thomas Nelson Publishers, Scotland, UK. s.v. Fadaei, M., 2015. Khorram and Ziba. Aria Land Publications, Tehran (in Persian). Feliks, J., 2006. Saffron. In: Encyclopedia of Judaica. s.v. Finkelstein, Silberman, 2015. The Archeology of the Bible (S. Kareempoor, Trans.). Sabzan Publications, Tehran (in Persian). Gouya, M.Kh, 1963. Divan-e Gooya. Munsh¯ı nuvil Kish¯ur, Tehran (in Persian). Hafez, M., divan, Tehran, Nashr Mohammad, 1371 SE/ 1992 CE. Hawkes, J., 1998. The Dictionary of the Bible. Asatir Press, Tehran. Hayyim, S., 1981. Hebrew-Persian Dictionary. Jewish Association of Tehran, Tehran. Hekmat, A.A., 1958. Indian Land. University of Tehran Press, Tehran (in Persian). Jahangir Gurkanid, N.M., 1980. Jahangir Nameh. Iranian Culture Foundation, Tehran (in Persian). Jones, A., 1990. Jones’ Dictionary of Old Testament. Kregel Publications, Grand Rapids, US. s.v. Kay Khosrow Esfandiar, 1983. The Elementary School of Religions. Tahoori Publications, Tehran (in Persian). Kermanshahi, A.A., 1993. Meraat Alahval Jahan Nama. Ansarian Publications, Qom (in Persian). Koleini, M.Y., 1401. Alkafi. Ali Akbar Ghafari, Beirut, Lebanon (in Arabic). Laleh Tik Chand, 2001. Bhar-e Ajam (Ajam Spring). Talaye Publications, Tehran. Lali Badakhshi, M., 1990. Samaratol Ghods. Scientific and Cultural Publications Company, Tehran (in Persian). Lewis, B., 1984. Jews of Islam. Princeton University Press, Princeton, NJ. Lo¨w, I., 1924. Die Flora Der Juden, II, Iridaceae-Papilionaceae. Leipzig University, Saxony, Germany. s.v. Masoudi, A.H., 1968. Morouj al-Zahab (A. Payandeh, Trans.). Translation and Publishing Agency, Tehran (in Arabic). Monir Lahoori, n.d. Masnavi in Traits of Bengal. Lahore, Karachi. Moslem Ibn Hajjaj Neyshabouri, 1954. Sahih. Dar ul-Ehya al-Toras al-Arabi, Beirut, Lebanon (in Arabic). Nagouri, Sh.A., 1993. Akbarnameh Ministry of Culture and Higher Education, Tehran (in Persian). Nomani, Sh, 1984. Sher al-Ajam (M.T. Fakhr-Daei-Gilani, Trans.). The World of Book Press, Tehran (in Persian). Onsori Balkhi, 1984. Divan-e Onsori Balkhi. Sanaee Publications, Tehran (in Persian). Perlman, M., 1965. Ghiyar. EI2, vol. 2. s.v. Rodriguez, H., 2006. Introducing Hinduism. Routledge Press, New York. Shooshtari, M.A.Kh, 1984. Tohfatol Alam. Tahoori Press, Tehran (in Persian). Swami, S.T., 2005. The Ayurveda Encyclopedia, Natural Secrets to Healing Prevention Longevity. Ayurveda Holistic Center Press, New York.

Further reading Muhammadifar, Sh, 2016. Saffron, Encyclopedia of the Islamic World, vol. 21. Encyclopedia Islamica Foundation, Tehran (in Persian). Salim Tehrani, M.Gh, 1970. The Complete Divan of Muhammad Gholi Salim Tehrani. Tehran, Ibn-Sina Press, Tehran (in Persian). Stein Sultz, A., 2004. The Talmud (B. Talebi-Darabi, Trans.). Research Center for Religious Studies, Qom (in Persian).

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Chapter 3

Saffron in the ancient history of Iran Askar Bahrami1, Sayed-Said Mirmohammadsadegh2, Seyyede-Fatemeh Zare-Hoseini3, Reza Mousavi-Tabari2 and Salman Saket4 1

Encyclopedia Islamica Fundation, Tehran, Iran, 2Written Heritage Research Center, Tehran, Iran, 3Department of Religion and Comparative

Mysticism, Faculty of Theology and Islamic Studies, Ferdowsi University of Mashhad, Mashhad, Iran, 4Department of Persian Language and Literature, Faculty of Letters and Humanities, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 3.1 Introduction 3.2 Saffron in Iran before Islam 3.2.1 Use of saffron before Islam 3.2.2 Use of saffron in ancient Iran 3.3 Saffron in Iran after Islam 3.3.1 Cultivation of saffron

3.1

23 23 24 25 28 28

3.3.2 Saffron as a commodity 3.3.3 Applications of saffron in Iranian daily life 3.4 Conclusion References Further reading

28 30 32 32 34

Introduction

Iran is a country whose territory was once not limited to its current borders. Large regions such as the Caucasus, West Asia, Central Asia, and parts of South Asia were parts of Greater Persia. Historically, these regions were ruled by various imperial Iranian dynasties such as Achaemenids, Partians, Sassanid, Samanids, Safavids, Afsharids, Zands, and Qajars. In recent centuries, Iran lost its vast territories and the boundaries of the country became limited to the present ones. Traditionally, Iranian history is divided into before and after Islam. In CE 651, with the death of Yazdegerd III, the last king of the Sassanid dynasty, the ancient Iranian history was over and the Islamic period of the Iranian history began. Based on this, the authors assess saffron production and use in two periods: before and after Islam.

3.2

Saffron in Iran before Islam

There is not much agreement on the origin of saffron; however, Iran has been introduced as one place of origin. There is little and scattered historical evidence on the cultivation and use of saffron and in the texts left from the ancient Iranian languages—namely Avestan and Old Persian, whose period of prevalence is considered to be the end of the Achaemenid era (330 BC)—this plant is not mentioned. However, other texts from the other branch of Indo-Iranian (i.e. Indians) tribe, at nearly the same time, have, though very little, evidence of this plant. Also, in the Vedic Sanskrit texts (in Upanishads ii.3.10), a compound term has been translated as “(saffron) garment” (Macdonell and Keith, 1912, p. 163). In addition, Aeschylus, the Greek playwright, mentions the saffron sandals of Darius Achaemenid when describing his soul in a play called “Iranians” (Collard, 2008, p. 20). Many of the historical sources have mistaken saffron for turmeric and safflower (Carthamus lanatus) and, as a result, make the pursuit of evidence related to saffron in historical contexts difficult. One of the Sanskrit names for saffron is k¯avera, which is often mistaken for k¯aver¯ı or turmeric (both derived from a commercial site, which is referred to as “chaveris” in geography by Ptolemy) (Laufer, 1919, p. 309). The misapplication of the word saffron is also reflected in other Persian dictionaries. The expression “korkom” is considered by Persian dictionaries to be equal to saffron (Dehkhoda, 2001) and some of its forms are used to address other plants such as turmeric (Dehkhoda, 2001). The name of this plant has been cited in Greek as both κρoκoς and ζαϕoρα (Oxford English-Greek Learner’s Dictionary, 2006, Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00003-4 © 2020 Elsevier Inc. All rights reserved.

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p. 616) where the former is related to korkom in terms of root and the latter to saffron. Ancient Chinese references attriˇ bute saffron not only to Kashmir but also to Iran in the Sasanian period. One of these sources (Cou sˇu) features the Yu¨-kin product as one of the products of the po-se country (Pars), which is also reported by Sui sˇu. In fact, saffron (Crocus), planted by Iranians, has existed as a self-growing plant for a long time (Laufer, 1919, p. 312). According to some researchers, saffron was never planted in India and probably was imported to Kashmir from Iran. In the Sanskrit sources, saffron has been referred to as kasmira and kumkuma, with the first term indicating the plant entered other parts of India from Kashmir and the other relating to the name Korkom indicating it at least entered India from Iran (Sensarma, 1992, No. 175, 210). However, the third Sanskrit name for saffron is v¯ahl¯ıka (Sensarma, 1992, No. 422), meaning “Balkhi, coming from Balkh,” which means pahlava or “part” (Laufer, 1916, p. 459). It should be noted that in Tibetan texts three types of saffron (for medical use), named based on their origins, are mentioned: the first one, K¯as´mira, came from Kashmir, the second one, V¯ahl¯ıka, Balkhi from Balkh, and the third one, P¯aras¯ıka, Parsi coming from Pars (Dash, 1976, pp. 62
63). The name of saffron can also be found in the Sogdian language, a language of the eastern middle and the mediator language of the Silk Road and throughout Central Asia. In Sogdian Buddhists texts, in which the word kwrkwnph has appeared, it has been translated as “Korkom Indian saffron” (Gharib, 2017, p. 201). There are more works concerning that topic in the writings of the Middle Persian (from about 330 BC to about CE 652). The Persian text of Bundahishn is the interpretive translation of part of Avesta text; however, its final version, which was compiled during the CE 9th century, contains a chapter on plant classification. In this part, saffron is introduced as karkom or korkom (Bahar, 1990; Farahvashi, 2011, p. 342; Mackenzie, 1971, p. 52; Pakzad, 2005, p. 213). Other forms of this term are found in most Semitic languages as well as Indian languages: Hebrew: kark¯om; Akkadian: kurk¯am and kurk˘am¯a; Syriac: k¯urkam¯a; Arabic: karkum; Indian: kunkum and kumkum; and Tamil: kunkumam (Kronfeld, 1892, p. 14). While some scholars believe the Pahlavi karkum or kalkum is a substitute for the Semitic languages (Bahar, 1990, p. 184), others cast doubt upon this origin (Laufer, 1919, p. 321). In a report in Bundahish (One of the most important works of Zoroastrian middle Persian language) on plant classification, kurkum is a kind of flower: "whatever has odoriferous blossoms, and grows in various seasons by the hand labor of men, or has perennial root, and blooms in its season with new shoots and sweet scented blossoms, such as the rose, the narcissus, the jasmine, the sweet-briar, the tulip, the colocynth, the pandanus, the “chapag,” the “kheyri,” the kurkum, the swallow-wort, the violet, the palm tree flower, and others of this kind they call the flower plant" (Dadagi, 1990, p. 87). Kurkum, in addition to other plants including alizarin and indigo, is a type of dye (Bahar, 1990, p. 88) and since every flower belongs to one of the Amesha Spentas (the first creatures of Ahura Mazda and his close friends) and other gods and goddesses, korkom is a relative of Maraspand or Mansaraspand (the Holy Word and a Zoroastrian goddesses) (Dadagi, 1990, p. 88).

3.2.1 Use of saffron before Islam It appears that the first saffron fields of the world were first established in the state of Mad (Hamadan and Holwan in Iran) (Abrishami, 2004, p. 239) and subsequently spread to Ray, Qom, Isfahan, and coastal areas of the Caspian Sea as well as the Pars state. Later, the cultivation of saffron became common in Transoxiana and Khorasan, and finally, the production of high-quality saffron became massively concentrated in Qohestan and Qaenat (Abrishami, 1997, p. 74). The production and trading of saffron has always been done by Iranians. The Iranians, while exporting saffron to many parts of the ancient world, introduced its properties to the Greeks, Romans, Chinese, and Sami people like the Arabs. In prehistoric paintings, saffron pigments, which were created in present Iraq and northwestern Iran 50,000 years ago, are found (Willard, 2002, p. 2). The Sumerians used self-growing saffron as the main substance in magical potions (Willard, 2002, p. 12). In ancient Iran during the 10th century BC saffron was cultivated in Darband, Isfahan, and Khorasan and its threads were placed in textiles (Willard, 2002, p. 2), which were bestowed upon the goddesses during rituals. Also, saffron itself was used to dye textiles and in perfume, medicine production, and bathing (Willard, 2002, pp. 17
18). Also, ancient Iranians used to treat melancholia by scattering saffron around the bed and mixing it with hot tea. However, non-Iranians feared the use of saffron as a medicinal and aphrodisiac substance by Iranians (Willard, 2002, p. 41). During the Sassanian era, saffron planting in Qom and Bavan (located in the district of Ganj Roosta midway between Herat to Sarakhs near Badghis) became popular, as is reflected in some of the texts of that time. In a Pahlavi treatise named Khosrow Ghobadan and Ridak, belonging to the late Sassanid era, which contains a conversation between Khosrow and Anushirvan, Khosrow Parviz and his ghulam (male servant) named Ridak, in response to

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Khosrow’s question about which flower or plant is more aromatic, Ridak mentions a number of flowers and plants without naming saffron. Nevertheless, in its Arabic translation, the saffrons of Qom and Bavan have been acclaimed and described as having heavenly scents (Al-Tha’alibi-Neyshabouri, 1993, p. 393). It is believed that the term was probably later added to the original Pahlavi text (Abrishami, 2004, pp. 131, 253).

3.2.2 Use of saffron in ancient Iran Most likely, ancient Persians, especially Zoroastrians, considered saffron a divine creature due to its positive properties, beautiful and eye-catching color, and delicious aroma because from the point of view of Persians, especially Zoroastrians, any good and beneficial being is created by Ahura Mazda. Considering the importance and various uses of saffron in ancient Iran, its location, properties, and applications can be summarized as follows.

3.2.2.1 Saffron for food applications During the Achaemenid era, saffron was widely used by cooks and crew to produce colorful and diverse dishes (Abrishami, 2004, p. 243). In this era, saffron was used in the combination of enlivening and restorative spices, as well as in the combination and decorating of fine cookies and some types of special taftoon bread (a kind of bread in Iran) (Justi, 1935, p. 26). It is said that Alexander the Great, influenced by Persian culture, traditions, and kings, used saffron in decoctions and rice (Willard, 2002, pp. 54
55). During the Greeks domination over Iran, they also became familiar with many native herbs of the Achaemenid territory including saffron. During the Parthian era, the same common foods of Achaemenid such as saffron were consumed (Abrishami, 2004, p. 250). In the Sassanid era, making saffron pastries, breads, and cookies was prevalent and wine was produced with saffron (Abrishami, 2004, p. 268); in addition, paloodeh (a traditional Iranian cold dessert) was a common sweet at that time (Abrishami, 2004, p. 277). In Shahnameh, in the story of Zahh¯ak, Satan, disguised as a young chef appears and provides Zahh¯ak with a beef dish that had been marinated with saffron (Khaleghi-Motlagh, 2007, vol. 1, pp. 49
50): On the fourth day, he (Zahhak) ¯ prepared food made from beef/ Marinated with saffron and golab ¯ (rose water) that is the aged wine and pure musk.

In the story of Bahram Gur, the merchant who was Bahram’s host, while preparing to cater for Bahram, goes to bazaar and buys saffron (Khaleghi-Motlagh, 2007, vol. 6, p. 465): He searched for sugar, almond, chicken and lamb to finalize the preparation of the feast/ He carried wine, saffron, musk and golab ¯ while going home with his heart filled with thrill.

3.2.2.2 Medical and pharmaceutical uses Saffron has long been known in Iran as an effective ingredient for the provision of medicines and for the treatment of various diseases. In particular, during the Sassanid era, it was used as a medicinal herb for oral treatments, especially in kings’ palaces (Najmabadi, 1962, pp. 416
417). Saffron even had some applications in battles. For example, Alexander would take a shower with saffron in the middle of his battles, and later the custom of bathing with saffron was followed by Alexander’s army and was taken to Greece (Willard, 2002, pp. 54
55). In the narrative related to the Shapur War, the son of Ardashir Sassanid, with the Zizan, the Arab Amir (king), it has been mentioned that Shapur intended to conquer the fort. Having seen Shapur from the top of the fort, Zizan’s daughter falls for him so passionately and as a result, betrays her father and devises a scheme and informs Shapur through a letter. Based on this scheme, the guards of this stronghold become drunk with “saffron wine”; consequently, Shapur reaches the city and Zayzen as well and kills him (Dinavari, 1991, p. 75). Saffron was also used for abortion. In ancient Iran, the abortive properties of some drugs were well known. One such drug named “shaeteh” was mentioned in Avesta (Avesta, 1991, pp. 14
15) and is comparable to saffron in terms of properties and is likely related to it. Moreover, as “shaeteh” was used for abortion, one of the main properties of saffron has appeared in old medical and pharmacological resources. It should be noted that the term “shiati” in ancient Persian and the term “shadih” in Sassanid Pahlavi both mean “joy” and are related to “shaeteh” on the one hand and the joyful and abortive properties of saffron on the other, while proving that requires more examination (Abrishami, 2004, p. 472).

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3.2.2.3 Cosmetic use In Iran, saffron was recognized as an aromatic substance and it was believed that burning it as well as applying its incense to fill spaces with fragrance. In addition, it was processed in various ways to aromatize and decorate the skin and keep it fresh. Using saffron as perfume and incense in Iran goes back to ancient times, based on historical evidence and documents (Abrishami, 2004, p. 375). Indeed, saffron is among the first tanacetum balsamitas and plants used to produce oily and watery perfumes and cosmetic oils (Abrishami, 1997, p. 247). In the Achaemenid era, saffron was used to aromatize the king’s throne and surroundings (Abrishami, 2004, p. 243). Ferdinand Justi, while describing the life of Darius Achaemenid, wrote: The Iranian king rubbed special fragrant oil over his body that consisted of a combination of sunflower oil cooked in milk fat, saffron and date wine. Justi (1935, p. 17).

During the Parthian period, the kings and magies (moghan) also made a mixture of herbs with milk fat, saffron, and date wine and used it as makeup for their faces (College, 1979, p. 81).

3.2.2.4 Ritual uses Saffron was also used in variety of ceremonies and rituals in ancient Iran with varied applications such as an incense, donating and sprinkling it mainly in rituals held to welcome elders, gifting it in weddings, and drinking saffron syrup in religious ceremonies. The following provides evidence of these applications. 3.2.2.4.1 Burning and incensing saffron This was common in ceremonies and celebrations and also to welcome elders and victors. Besides using incense to give palaces, rooms, and surroundings a pleasant fragrance, it had therapeutic and disinfecting benefits too (Abrishami, 1997, p. 251, 253). In Shahnameh, it has been mentioned that Fereydun, after holding a light ruby glass of wine in hand, “He ordered to build a fire and they burned ambergris and saffron” (Khaleghi-Motlagh, 2007, vol. 1, p. 89). Saffron was burned at important ceremonies such as the interment of Rustam. According to Ferdowsi’s account, the undertakers burned ambergris and saffron after cleansing his wounds and drying them (Khaleghi-Motlagh, 2007, vol. 5, pp. 458
459). 3.2.2.4.2

Donating and gifting saffron

In ceremonies held to welcome elites such as kings, elders, grand people, and champions, musk, dinar, dirham (silver coin, the currency of Islamic world), and saffron were donated and thrown to receive such people and welcome them. In joyful ceremonies and celebrations, saffron was used to aromatize the atmosphere, and in fact, the strong aroma of saffron and the glittering of gold stimulated the brain and staring of eyes. Saffron donation and dirham and the burning of incense, ambergris, and saffron was done in winter (Abrishami, 1997, p. 255). In Shahnameh, when Iranians welcomed Bahram Gur returning from Kannuj it was noted that people (KhaleghiMotlagh, 2007, vol. 6, p. 595): Dirhams were thrown from horizon to horizon/ as well as musk, dinar and saffron.

When Kay Khosrow comes back to Iran from Turan, Iranians decorate the city and throw dirham, saffron, dinar, and musk (Khaleghi-Motlagh, 2007, vol. 2, p. 453): Once King Kay Khosrow returned to the city/ the world was filled with fragrance, colors and images. Thanks to the decoration, the world was decorated/ doors, roofs and walls were filled with ambitions. The musicians sitting everywhere/ golab, ¯ wine, musk and saffron.

According to Shahnameh, in some ceremonies during ancient Iran, similar to Persian diba, gem, gold, silver, and turquoise, saffron was also among precious and aristocratic gifts that were gifted. In particular, in engagement ceremonies and weddings, saffron was one of the gifts wrapped up and gifted. For instance, when Siavash gets engaged to Farangis, Golshahrbanoo, the wife of Piranviseh prepares 100 trays of saffron and sends them to Farangis (Khaleghi-Motlagh, 2007, vol. 2, pp. 302
303): Thirty camels were carrying gold, silver, trays and Persian clothes

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One hundred trays of saffron and musk were carried, along with Golshahr and her sisters.

Among the gifts Manuchehr gives to Sam, in addition to the Arab horses decorated with gold saddles, Indian swords with golden scabbards, dinar, fur, ruby, Roman slaves, trays of peridot, turquoise cups, crimson gold, and raw silver covered with musk, camphor, and saffron are seen (Khaleghi-Motlagh, 2007, vol. 1, p. 177): The trays of peridot and turquoise cups/ were either crimson gold or raw silver. Full of musk, camphor and saffron/ were all brought by obedients.

3.2.2.4.3 Decoration of steeds with saffron In addition to decorating festivities with fragrant substances, occasionally kings, elders, and champions would decorate their elephants and horses using musk, wine, gol¯ab, and saffron. This was also done to the forelocks and manes of S¯am and Z¯al’s horses on the day of their arrival to Kabul to visit Rudaba (Khaleghi-Motlagh, 2007, vol. 1, p. 262): All horses’ manes and forelocks were covered with musk and saffron.

On the day Rostam triumphantly returned from the battle with Afrasiab, an elephant, the face of which was covered with musk, wine, and saffron, was brought to him at Kay Khosrow’s command (Khaleghi-Motlagh, 2007, vol. 3, p. 281): The entire elephants’ mane was filled with musk, wine and saffron from horizon to horizon Countless of saffron and dirham were thrown and musk and ambergris were sifted too.

The mane of Siavashe’s horses were decorated with musk, wine, and saffron on the day of his visiting with Kavous (Khaleghi-Motlagh, 2007, vol. 2, p. 208): The horse’s mane covered with musk, wine and saffron.

3.2.2.4.4

Drinking saffron syrup

Using saffron syrup (which was a mixture of gol¯ab, sugar, water, ice, basil, and saffron) was common in some Zoroastrian ceremonies and rituals (Razi, 2010, p. 263). 3.2.2.4.5 Magical uses According to the evidence, saffron had special magical and pseudomagical applications such as in various amulets for the exit of the jinn and evil spirits from the body of the infected. In one of Zoroastrian texts called The Narrative of Darab Hormozdiar in the Pahlavi language (Unwala, 1922, vol. 2, p. 275) about preparing a kind of amulet used to relieve headache, it was mentioned that after crushing musk and saffron and mixing them with wine, a substance was obtained with which “nirang” (a spell or script similar to prayer used to eliminate problems and illnesses) had to be written on a deer’s hide and worn on the left arm. Also, it was mentioned that if on the day of Esfandarmaz (Esfand fifth), when a celebration was held, a nirang was written with saffron on deer’s skin or paper and then put on the roof, no illness would strike that area in that year (Unwala, 1922, vol. 1, p. 527). 3.2.2.4.6

Writing uses

Surprisingly, the use of saffron as ink in ancient Iran goes back to the mythological era, likewise it is said that at the time of Garshasp, one of mythological Iranian heroes, using saffron and musk as ink was common (Yaghmaie, 1975, p. 433). In Sassanid era, hides were covered with saffron so that the unpleasant smell would disappear. It is said that because Khusrow Parviz had a delicate nature and disliked the bad smell of hides used for writing, he ordered that no secretary was allowed to send him the tax lists unless they were written on hides that had been covered with saffron and gol¯ab (Balazori, 1988, pp. 368
369). 3.2.2.4.7 Applications of saffron in astronomy and astrology In an ancient astronomical text called Tang Loosha (Homayoon-Farokh, 1987) written in Sassanid Pahlavi, for each day of the year, a celestial range and descriptive chart is presented and the horoscope of the people born on that day determined. To describe the horoscopes of the people born at these times (the 19th degree of Cancer, the 23rd of Libra, and

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the 16th of Pisces) the author mentions the pleasant aroma of saffron and predicts a bright future (Homayoon-Farokh, 1987, pp. 67, 275, 123, 293, 248, 325).

3.3

Saffron in Iran after Islam

The earliest reports of the cultivation of saffron in Iran in the Islamic period date back to the 9th and 10th centuries AH, based on which a vast territory from east to west and from southeast to northwest of Iran can be regarded as saffron cultivation areas. For example, in the northeast of Iran in Vararud, around Tirmidh; in the east in Qahestan, Ghaen, Birjand, and Torbat-e-Hydarieh; in the southeast in the city of Daghash of Zamindavar lands of Sistan; in the central regions in Hamedan, Nahavand, Qom, Rey, and Isfahan; in west and northwest in Soltanieh, Tabriz, and Baku; and in the south in Gur (Firouzabad), Estehbanat, and Shiraz. There are also many villages in different regions of Iran named with derivatives of the word saffron in Persian such as Zaferan, Zaferani, and Zaferanieh, which indicates the vast range of saffron cultivation and production throughout Iran. But according to the available evidence, the range of cultivation and production of saffron was gradually reduced from the late CE 19th century, so that from the Qajar period its cultivation was limited to the cities of Khorasan.

3.3.1 Cultivation of saffron Saffron is planted in most warm and cold climates, but it yields better in cold climates. The time of planting is in the summer. After plowing the land with a shovel, the seeds are spread and covered with soil. The land should not be irrigated for up to 40 days. In the first year, the crop yields few flowers. Saffron plants live between 3 and 5 years on a land. Mice are the main pests of saffron, and when the leaves go yellow, it is clear that its root has been eaten under the ground (Sotoudeh and Afshar, 1989, pp. 203
204). The planting distance of saffron bulbs from each other is a span (about 22.5 cm). The best time to harvest saffron flowers is in the early morning, when the flower forms a bud; the head of the bud will be held straight and the end is taken out of the bulb (Moshiri, 1967, pp. 210
211). Lamei-Gorgani, a 5th century AH poet, refers to the season of flowering and harvesting of saffron in the fall: I am like a strand of saffron in fall/ She is like a bunch of Jasmines in the month of Nisan. Dabir-Siaghi (1976, p. 109)

Or: As the fall comes, the blessed Mehregan is upon us/ Flowers flourish, saffron flowers, bergamot orange comes, and basil grows. Dabir-Siaghi (1976, p. 138)

3.3.2 Saffron as a commodity The first narrations of zakat (a form of alms-giving treated in Islam as a religious obligation or tax) on saffron show that the value of this plant was not a fixed one. According to the book Fotuh-al-Boldan, the scholars of Hijaz disagreed on the amount of zakat payment on saffron, as it has been quoted in their arguments. But some scholars believed that zakat applies to saffron, while others limited the matter to the power of land, and some did not believe in the necessity of paying tax for this product (Balazori, 1988, p. 108). Perhaps this disagreement arises from the lack of proper knowledge on the use of this valuable plant. However, historical documents from the Islamic era show that saffron has always been among the agricultural products of higher value, and according to Rashid al-Din Fazlullah Hamedani (Sotoudeh and Afshar, 1989, p. 205): “It is easy to consume but it is very useful, therefore, it’s a fine product which is not found in any land and it costs a fortune.” An examination of existing historical documents shows that the amount of tax demanded from saffron was higher than that of other plants; for example, in the CE 10th century in Qom, even the tax taken from a destroyed land of saffron was calculated to be half the amount of an arable land (Tehrani, 1982, p. 108). The oldest information on the taxes taken from saffron is for the saffron of Nahavand, which was 30 dirham per acre (Tehrani, 1982, p. 120). After that, there is information of taxation in Tabarestan, where in the days of Mansour Davaniqi, the

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Abbasid Caliph, Esfahbod Khorshid, the ruler of Tabaristan, in addition to 300,000 dirhams, and several other valuable goods, sent 10 kharvars (ass-loads; Kharvar is the unit of weight, equal around 300 kg) of saffron to Baghdad, which according to Ibn-Esfandiar, nothing like that had happened in the whole world (Eqbal, 1941, vol. 1, p. 175). There is no information on the taxation in other cities except Qom; taxation of saffron in Qom in the CE 10th century was in all the villages of Qomroud, except Tabrash (Tafresh) Dakhel, Jast, and Faleq, and was 62 Dirham for each acre, and in Tabresh Dakhel, Jast, and Faleq, it was 43 dirhams and one-third for each acre (Tehrani, 1982, pp. 121
122). There is other information on the taxation of saffron from Qomi historians from CE 1668 about the wage given to land surveyors and the people who determined the taxation (Modarresi-Tabatabaie, 1975, p. 245); this report shows that at that time, saffron was being cultivated in Qom. The oldest price rate of saffron goes back to the time of Karim Khan Zand, when each shahi maund of saffron was worth 25,600 dinars (Moshiri, 1978, p. 315). Given the prices available from this commodity in the Qajar period, it can be said that the price of saffron was on the rise in this historical period, and each mithqal of saffron cost 20 dinars between the years of 1153 and 1163 AH, while it rose to 120 dinars during Nasir al-Din Shah Qajar (Abrishami, 1997, p. 601). In the year 1924, the price of saffron in Birjand, which was sold in Isfahan, was 835 Rials per kilo (Nasr, 1992, p. 166). Historical, poetic, and allegorical evidence also shows that saffron was an expensive commodity. Najmuddin Abu al-Raja Qomi refers to saffron as wealth and gold coin. When he speaks about the ministry of Kamal al-Din Mohammad Khazan, he writes that after him and Nizam al-Mulk, “there were many high-ranking ministers who wasted oodles of saffron by their lack of tact” (Daneshpazhouh, 1984, p. 100). Saffron was also a valuable commodity rulers sent around to neighbors as tributes (Ghaffari-Fard, 2004, p. 701). Manouchehri-Damghani (died in CE 1041) implicitly refers to the high price of saffron in a verse (Dabir-Siaghi, 1968, p. 25): If my face gets jaundiced for being a lover, it’s all the same/ As saffron is even dearer than red tulip.

Ghatran-Tabrizi also compares the yellowness of his face and the sanguine face of the beloved, and says that “despite being as yellow as saffron, I am as worthy as tulip which is abundant and cheap, while despite the similarity of the face of beloved to red tulip, she is as dear as saffron” (Nakhjavani, 1983, p. 313): I have the color of saffron, and she is like the tulip petal/ I am as cheap as tulip and she is as dear as saffron.

But Amir Khosrow Dehlavi is happy to buy the soil of the beloved’s residence at the price of saffron (Nafisi, 1982, p. 72): I place my face on your doorway, and If you allow me/ I will buy its soil at the price of saffron.

Among the poems of Naser Khosrow (died in 471 or 481 AH), there is a verse indicating that some merchants sold fake saffron due to the high cost of it: For your duplicity, you do not care, if you pass off hay as saffron.

According to Naser Khosrow, fraudsters used Reseda to make counterfeit saffron (Minavi and Mohaghegh, 1978, pp. 14, 199). Khaghani (died in 595 AH) also refers to cheating on the sale of saffron by filing and drying the cooked beef (Sajjadi, 1979, p. 85): Wherever there is a companion, there are also opponents, Yes, the load of saffron is beef.

In the 7th century AH, Ibn-Ikhwah noted the addition of chicken meat to saffron as well as beef. To identify fake saffron, he said that “soak a little of saffron in vinegar, if the strands attached to each other and were combined, it is adulterated and counterfeit; otherwise, it is pure” (Ibn-Ikhwah, 1968, p. 122).

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In proverbs, the high price of saffron is mentioned. Perhaps the oldest phrase used in this regard is in Tarikh-i Jahangushay written by Juvaini. In a quoted story from Qa’an, Juvaini points out that Moukakhatoon, Qa’an’s wife, wished to give her two pearl earrings to the person who brought a couple of melons. In order to make his wife understand that the melon seller does not know the price of the earring, Qa’an uses the phrase: “like saffron before a longeared donkey” (Qazvini, 2004, vol. 1, p. 168). Qadri-Shirazi in the Safavid era uses a turn of phrase that indicates the scarcity and the price of commodities in which the price of hay is equal to saffron (Kheirandish and Vosoughi, 2005, p. 106). Ghasemi-Kermani, a poet of the Qajar period, also has an interesting turn of phrase for the pricing of goods by some people, and in his poem he says that if saffron is priced by donkeys, the price of it will be the same as hay (Afshar, 1993, p. 118).

3.3.3 Applications of saffron in Iranian daily life Saffron has different applications in Iranian daily life. For most contemporary Iranian people, saffron is used in food. However, historically, in addition to its function as food, this valuable plant provided odor and aroma and was even used in dyeing despite its high cost; it was also known for its medicinal properties. The medical applications of saffron are discussed extensively in other chapters of this book, so in this section we will focus on the other three uses.

3.3.3.1 Odor and aroma Saffron is one of the most famous fragrances and odors (Imam, 1963, p. 307; Minavi and Harirchi, 1976, pp. 125, 302
303; Sajjadi, 1966, p. 45) used in religious environments (Homayoon-Farokh, 1987, pp. 67, 248, 325), and has been used together with musk, amber, and camphor to create incense. According to one report, Mehdi Abbasi ordered the walls of the House of God to be covered with musk and saffron (Mohaddes, 1993, p. 93). Also, Hesam al-Dawla Ardeshir Bavandi, son of Esfahbod Hassan, from Espahbodan of Tabarestan, burned saffron in golden censors in his castle, together with agarwood (aloes-wood or oud) and ambergris (anbar) (Eqbal, 1941, vol. 2, p. 121). Saffron, in addition to odor, has also been used to make perfume. Shahmardan Abil-khair in Nezhat Nameh Alaei mentions several perfumes, one of the ingredients of which was saffron (Jahanpour, 1983, pp. 546
547). Saffron was one of the fragrances women used in their makeup (Homayoon-Farokh, 1987, pp. 123, 293). Due to the fact that saffron is warm and light (Afshar, 1967, p. 226), its aroma must also be considered a warm-tempered smell.

3.3.3.2 Dyeing In ancient times, both in medical and nonmedical books, yellow is interpreted as saffron. In the coloring of wool and fleck of carpets, textiles, and even paper, it is also referred to as saffron color (Zakaria-Kermani, 2009, p. 64). However, given that saffron was expensive, dyers often made yellow with turmeric, sumac, dried pomegranate skin, snapdragon leaves, berry leaves, reseda, pepper, and safflower, which is known as dyeing saffron (Baker, 2006, p. 33; Zakaria-Kermani, 2009, pp. 55
57). Despite its high price, dyers used saffron in dyeing by diluting it; this expensive color was used more for expensive silk fibers (Baker, 2006, pp. 33). Saffron was also used to make gold plating (Dimand, 1986, pp. 77
85). Before the gold plating, the plate was colored with saffron (De´roche, 2000, p. 158). Due to the solubility of saffron extract in water, lack of need for adhesives, and its transparency, it was used in coloring epigraphs, titles of Qur’anic Surahs, chapter headings, and some letters. Saffron extract was also used to reduce the solution acidity of colors such as rust and paper corrosion (Barkeshli, 1998; Mayel-Heravi, 1993, pp. 530, 566). In particular, saffron has been used in the Persian miniature paintings since the 13th century as an inhibitor of destructive verdigris (zangar) effects due to its pH stability (Barkeshli, 2018; Barkashli and Atale, 2002). The inhibitory effect of saffron on the destructive effects of verdigris has even been reflected in Persian texts in the form of mystical poetry (Barkashli, 2013, 2015). Saffron is also used to create a variety of colors such as russet, cathay, yellow, golden, pistachio, rose, and orange. Another use of saffron is in producing ink (Keshmiri, 2017, p. 160; Mayel-Heravi, 1993, p. 555). Saffron ink is used for writing, painting, and illustrating (Mayel-Heravi, 1993, p. 96, 156). Motavvas ink, from the Middle Ages, is made of saffron; it appeared to be colorful like peacock feathers or what today is referred to as luminescent (Mayel-Heravi, 1993, p. 96, 97, 192). Another use of saffron was for starching paper (Noshahi, 2001). In addition, the color of saffron is used by Persian-speaking poets to describe jaundice of the face, sadness, and sometimes as a sign of asceticism and austerity.

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You show up and changes occur again, My face gets yellow like saffron for your temper. Adib-Boroumand and Nasiri-Kahnamouei (2005, p. 556) Make your face like saffron yellow by staying awake at night, So that your face is sanguine on the day of judgment. Afshari and Emami (2013, p. 726)

But more often than not, poets the term to describe fear, as when the face goes pale and saffron-like before an aweinspiring king: When he is in a fight wielding sword, spear and an iron helmet, the face of riders get like saffron in fear. Nakhjavani (1983, p. 481) Due to your lotus-like sword, from which water drips, The face of rebels goes yellow like saffron. Nourian (1985, p. 163)

3.3.3.3 Food The most important application of saffron in the culture and history of people in Iran is in food. An examination of ancient culinary and edible texts suggests that this valuable plant was used both in dishes and in drinks; also, due to its color, it was used to color and decorate food, and also used to give an appetizing taste. One of the most important pieces of evidence on the nutritional value of saffron in ancient Persian and Arabic texts is its application as a seasoning to give aroma to food (Imam, 1973, p. 61) and make it digestible and tasty (Behnamir, 1967, p. 289). Perhaps the oldest report on the use of saffron as a seasoning in Iranian history after Islam can be found in the book History of Qom, which mentions cooked meat marinated with saffron and cinnamon (Tehrani, 1982, p. 247). Saffron was also used in Iranian food such chickpea dishes now known Abgusht. The oldest mention of the use of saffron in chickpea dishes is in a book written by Safi (Golchin-Ma’ani, 1983, p. 414). Following him, BoshaghAtameh delicalely notes the saffron flavor: If you want chickpea dish to benefit you, Use herbal distillates with saffron therein. Rastegar-Fasaei (2003, p. 18)

In addition to varieties of ash, saffron is one of the additives in most of the ash dishes mentioned in ancient texts (Shafiee Kadkani, 1988, p. 66; Ashpazbashi, 1974, pp. 76
77); in addition to ash, saffron was an important flavoring and seasoning in stews, kebabs, Kuku, dolma, kadu bouranee, samosa, and some breads such as komaj (Afshar, 2007, 1981a,b; Golchin-Ma’ani, 1983, p. 414; Mojahed, 2007). Saffron was also used to color food. The most famous coloring use of saffron is in saffron pilau, which is mentioned in ancient texts by many references. In addition to saffron pilau, in foods made with rice with meat, chicken and fish, and also in combination with legumes, depending on the type of pilau, saffron was used to color the food (Afshar, 2007, 1981a,b; Ashpazbashi, 1974; Mojahed, 2007; Olivier, 1992, p. 166). Saffron was also used to decorate food in Iranian cuisine. Perhaps the oldest reference in which mentioning to the use of decorating food with saffron is the Collection of Athir al-Din Akhsikati (died in CE 1181), which is to decorate Ash Zireh-ba with saffron: Porch becomes yellow with the rays of sunrise, Like Ash Zireh-ba that gets yellow with saffron. Farrokh (1958, p. 258)

Apart from ash dishes, the side dishes were also decorated with saffron because it made food look good (Afshar, 1981a,b; Mojahed, 2007, p. 34).

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Saffron has a very colorful effect on desserts Iranians serve after the main course. Among them, are different kinds of halvahs (gozab khorma (Yousefi, 1971, p. 101), yellow flower, starch, common purslane, carrot, vervain, tobacco (Afshar, 2007; Golchin-Ma’ani, 1983, p. 414; Ashpazbashi, 1974, pp. 48
49), where saffron is a flavoring and a colorant; in addition to halvahs, saffron has had numerous applications in the preparation of Sholeh Zard (Ashpazbashi, 1974, p. 77; Molavi, 1990, pp. 196, 339) and in sweets such as loz (Afshar, 2007, pp. 5, 51), zolbia (Moshiri, 1967, p. 259), sohan (Hakim-Souri, 1939, vol. 1, p. 25), also saffron syrup (Hakim-Souri, 1939, vol. 2, p. 21; Sefatgol, 2007, p. 167), and saffron distillate (Ibn-Hawqal, 1987, p. 65; Khazarji, 1975, p. 80). Saffron is also considered important because of its exhilarating effect. In ancient times, even looking at a saffron farm was considered exhilarating (Dabir-Siaghi, 1957). However, the effect of the saffron farm may be due to its smell. Saffron pickers and those who use saffron in their food are not exempt from its vibrant and exhilarating effects. In the following there are some examples from early and later poets on this matter. It is worth noting that poets considered yellow as the color of sadness, which is in conflict with the exhilarating nature of saffron, and the question has been cast in many ways by poets: If saffron makes one exhilarated, Then why does flowering of saffron make the flower gardens and gardens sad? Eqbal (1939, p. 513)

This subject was also mentioned in the following verse in Divan of Khaghani: If one only gets happy by saffron, It shows his heart is as sad as saffron color. Sajjadi (2003, p. 35)

And Seyed-Hassan Ghaznavi says: If saffron makes on smile, Why has crying made my face yellow as saffron. Modarres-Razavi (1983, p. 253)

According to historical records, there was a place called Zaferanja (place of saffron) that is naturally expected to be vivacious, but the Shiites gathered and mourned for Imam Hussein (AS), who was martyred in Karbala, in this site (Mohaddes-Ormavi, 2012, p. 406). Qavami-Razi, a Persian classical poet, refers to this contrast and conflict in a poem written as elegy for the martyrdom of Imam Hossein (AS): Tears come to the eyes in Zaferanja, The saffron, which brings smile. Hosseini-Ormavi (1955, p. 16)

3.4

Conclusion

The historical texts of Iran, the most important country in terms of saffron, show that although today saffron is cultivated in the eastern areas of Iran, it was grown in different parts of Iran previously. Reviewing these texts also shows that saffron was used in various ways throughout Iranian history and people used it in their foods, medical agents, etc. Comparing the different applications of saffron in different countries should be a topic of future research.

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66. Dehkhoda, A.A., 2001. Loghatnameh. Dehkhoda Dictionary Institution, Tehran (in Persian). De´roche, F., 2000. Manuel de codicologie des manuscrits en e´criture arabe. Bibliothe`que Nationale de France, Paris. Dimand, S.M., 1986. Islamic Industries Guide, (A. Faryar, Trans.). Publication of Scientific and Cultural, Tehran (in Persian). Dinavari, A., 1991. Akhbar al-Teval. (M. Mahdavi-Damghani, Trans.), third ed. Ney Publication, Tehran (in Persian). Eqbal, A. (Ed.), 1939. Divan of Amir Moezzi. Islamic Bookstore Publications, Tehran (in Persian). Eqbal, A. (Ed.), 1941. Tarikh-e Tabarestan. Khavar Publication, Tehran (in Persian). Farahvashi, B., 2011. Pahlavi-Persian Dictionary. University of Tehran Press, Tehran. Farrokh, R.H. (Ed.), 1958. Divan of Asir Akhsikati. Roodaki Press, Tehran (in Persian). Ghaffari-Fard A. Gh, (Ed), Tarikh-e Rashidi, 2004, Miras Maktoob Publication, Tehran, (in Persian). Gharib, B., 2017. Soghdi Dictionary, (Soghdi, Persian, English). Farhangan Press, Tehran. Golchin-Ma’ani, A. (Ed.), 1983. Lataef al-Tawayef. Eqbal Publication, Tehran (in Persian). Hakim-Souri, T.D.T., 1939. Koliat. Iqbal Press, Tehran (in Persian). Homayoon-Farokh, R. (Ed.), 1987. Tang Loosha (Sovar Dorj). Iranian National University, Tehran (in Persian). Hosseini-Ormavi, M.J. (Ed.), 1955. Divan of Ghavami Razi. Sepehr Publication, Tehran (in Persian). Ibn-Hawqal, M.A., 1987. Ibn-Hawqal’s Travelogue, (J. Sho’ar, Trans.). Amirkabir Publication, Tehran (in Persian). Ibn-Ikhwah, M., 1968. Aeen-e shahrdari, (J. Sho’ar, Trans.). Iranian Culture Foundation Press, Tehran (in Persian). Imam, S.M.K. (Ed.), 1963. Moqadamat al-Adab. University of Tehran Press, Tehran (in Persian). Imam, S.M.K. (Ed.), 1973. Ketab al-Tanvir. Nouriani Publication, Tehran (in Persian). Jahanpour, F. (Ed.), 1983. Nezhatnameh Alaei. Institute for Cultural Studies and Research, Tehran (in Persian). Justi, F., 1935. A Day in Dariush’s Life, (Sh. Rezazadeh, Trans.). Publication of Maaref Commission, Tehran (in Persian). Keshmiri, M., 2017. Illumination in Iran. Organization for the Study and Compilation of Humanity Field Books of Universities (SAMT), Tehran (in Persian). Khaleghi-Motlagh, J. (Ed.), 2007. Shahnameh. The Center for Great Islamic Encyclopedia, Tehran (in Persian). Khazarji, A., 1975. Travelogue of Abu dolaf in Iran, (S.A. Tabatabai, Trans.). Zavvar Publications, Tehran (in Persian). Kheirandish, A.R., Vosoughi, M.B. (Eds.), 2005. Jangnameh Keshm and Jerunnameh. Miras Maktoob Publication, Tehran (in Persian). Kronfeld, M., 1892. Geschichte des Safrans (Crocus sativus L. var. culta autumnalis) und seiner Cultur in Europa. Moritz Perles, Wien. Laufer, B., 1916. Loan-words in Tibetan. T’oung Pao 17 (4
5), 403
552. Laufer, B., 1919. Sino-Iranica. Chinese Contributions to the History of Civilization in Ancient Iran, Chicago. Macdonell, A.A., Keith, A.B., 1912. Vedic Index of Names and Subjects, vol. 1. Motilal Banarsidass Publisher, London. Mackenzie, D.N., 1971. A Concise Pahlavi Dictionary. Routledge, London. Mayel-Heravi, N., 1993. Book Illustration in Islamic Civilization. Astan Quds Razavi Publications, Mashhad (in Persian). Minavi, M., Harirchi, F. (Eds.), 1976. Al-Bolqah. Iranian Culture Foundation Press, Tehran. Minavi, M., Mohaghegh, M. (Eds.), 1978. Divan of Naser Khosrow. University of Tehran Press, Tehran (in Persian). Modarres-Razavi, M.T. (Ed.), 1983. Divan of Qaznavi. second ed. Asatir Press, Tehran (in Persian).

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SECTION | I Cultural and historical aspects of saffron

Modarresi-Tabatabaie, H. (Ed.), 1975. Khulasa al-Buldan. Hekmat Publication, Qom (in Persian). Mohaddes, M.H. (Ed.), 1993. Ma’athir al-Muluk. Rasa Press, Tehran (in Persian). Mohaddes-Ormavi, M.J. (Ed.), 2012. Al-Naqdh. Dar al-Hadith Publications, Qom (in Persian). Mojahed, A. (Ed.), 2007. Persian Food. University of Tehran Press, Tehran (in Persian). Molavi, M., 1990. Fih Ma Fih, Badi al-Zaman Foruzanfar. Amirkabir Publication, Tehran (in Persian). Moshiri, M. (Ed.), 1967. Ershad Al-Zeraah. Amirkabir Publication, Tehran (in Persian). Moshiri, M. (Ed.), 1978. Rostam al-Tawirakh. Amirkabir Publication, Tehran (in Persian). Nafisi, S. (Ed.), 1982. Divan of Amir Khosrow Dehlavi. Javidan Publications, Tehran (in Persian). Najmabadi, M., 1962. The History of Iranian Medicine. Honarbakhsh Publications, Tehran (in Persian). Nakhjavani, M. (Ed.), 1983. Divan of Ghatran Tabrizi. Ghoghnoos publication, Tehran (in Persian). Nasr, A. (Ed.), 1992. Al-Asfahan. Golha Press, Isfahan (in Persian). Noshahi, A., 2001. Bayaz-e khoshbouei. Baharestan Lett. 2 (4), 79
86 (in Persian). Nourian, M. (Ed.), 1985. Divan of Masoud sa’ad-Salman. Kamal Press, Isfahan (in Persian). Olivier, G.A., 1992. Travelogue, (Gh. Varahram, Trans.). Etelaat Publications, Tehran (in Persian). Oxford English-Greek Learner’s Dictionary, 2006. Oxford University Press, England. Pakzad, F. (Ed.), 2005. Bundahiˇsn: Zoroastrische Kosmogonie und Kosmologie. Centre for the Great Islamic Encyclopaedia, Tehran. Qazvini, M. (Ed.), 2004. Tarikh-e Jahangusha. Donya-ye Ketab, Tehran (in Persian). Rastegar-Fasaei, M. (Ed.), 2003. Koliat-e Boshagh-Atame. Miras Maktoob Publication, Tehran (in Persian). Razi, H., 2010. Gahanbars and Forudag Celebrations. Bahjat Publications, Tehran (in Persian). Sajjadi, S.J. (Ed.), 1966. Al-Merqat. Iranian Culture Foundation Press, Tehran (in Persian). Sajjadi, S.Z. (Ed.), 1979. Divan of Khaghani Shervani. Zavvar Press, Tehran (in Persian). Sajjadi, S.Z. (Ed.), 2003. Divan of Khaghani Sherwani. Zavvar Press, Tehran (in Persian). Sefatgol, M. (Ed.), 2007. Shajarat al-Muluk. Miras Maktoob Publications, Tehran. Sensarma, P., 1992. Plant names
Sanskrit and Latin. Anc. Sci. Life 12 (1
2), 201
220. Shafiee Kadkani M.R., (Ed), Halat wa Sokhanan-e Abu Saeed Abu al-Khair, 1988, Agah, Tehran, (in Persian). Sotoudeh, M., Afshar, I. (Eds.), 1989. Athar wa Ahia [Works and Revival]. Islamic Studies Institute of McGill, Tehran (in Persian). Tehrani, S.J. (Ed.), 1982. Tarikh-e Qom [History of Qom]. Toos Publications, Tehran (in Persian). Unwala, R., 1922. Narrations Darbnameh of Hormozdiar. Bombay (in Persian). Willard, P., 2002. Secrets of Saffron: The Vagabond Life of theWorld’s Most Seductive Spice. Beacon Press, Boston, MA. Yaghmaie, H. (Ed.), 1975. Garshaspname. Tahoori Publication, Tehran (in Persian). Yousefi, G.H. (Ed.), 1971. Taqvim al-Sihhah. Iranian Culture Foundation Press, Tehran (in Persian). Zakaria-Kermani, I., 2009. Termeh Shawls of Kerman. Art Academy, Tehran (in Persian).

Further reading Moein, M. (Ed.), 1997. Borhan-e Qate. Amirkabir Publication, Tehran (in Persian). Shafiee-Kadkani, M.R. (Ed.), 1988. Halat wa Sokhanan-e Abu Saeed Abu al-Khair. Agah Press, Tehran (in Persian).

Section II

Safron production

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Chapter 4

Evolution and botany of saffron (Crocus sativus L.) and allied species Mohammad-Hassan Rashed-Mohassel Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9

4.1

Introduction Crocus Contractile roots Corm Iridaceae Saffron evolution and phylogeny Pollination and seed growth Sexual reproduction Cultivated saffron (Crocus sativus L.)

37 38 40 41 41 43 46 46 47

4.10 Wild saffron (C. cartwrightianus Herbert) 4.11 Crocus palasii subsp. Haussknechtii 4.12 Crocus oreocreticus Burtt 4.13 Crocus Thomasii Tenore 4.14 Crocus hadriaticus 4.15 Conclusion Acknowledgment References Further reading

51 52 53 54 54 55 56 56 57

Introduction

Saffron spice refers to the desiccated stigma of Crocus sativus L and is the most expensive spice in the world. It is named “Red gold” and appreciated because of its color, flavor, and aroma (Lopez-Corcoles et al., 2015). Saffron is a triploid plant, propagated by corm (Fiore et al., 2010). The beauties of this small geophyte have resulted in generations of children’s stories, which generally include few facts. Based on the Julius brands documentation, the coplayer of Otared or Hermes was a young slave. When the slave’s blood was shed on the plain, saffron emerged. Abu-Raihan (an ancient Iranian scholar), without naming the slave, says Jalinnus had a slave that was killed and whose blood incorporated with the soil, and thus the saffron plant emerged. Zaryabkhouei (Abrishami, 2004) said the name of that young fellow playing with Hermes was Krocus, the Greek word for saffron. Pierre Grimily states Krocus was a young fellow who fell in love with similax. Assuming the aforementioned slave was Karkom, Karkimas, or Karkomiseein, in Persian dialogue it would be zaffran and in Greek, Krocus. It is probable that a story from Shahnameh Ferdowsi was delivered to Greece. In this story Siavash, the son of Kaykavous the king of Iran, was in love with Farangis, the daughter of Afrasiab the king of Tourane. Siavash was unfairly killed by Garsivaz, and where his blood poured out, Khoon-eSiavash (the blood of Siavash), a plant grew (Abrishami, 2004). This story may have spread to Greece and developed differently (Abrishami, 2004). A Greek story says saffron was so beautiful that Zeus slept on a bed of saffron (Grilli Caiola, 2010). Alexander the great and his army during his campaign in Kashmir stopped in a plain. The next morning he saw violet flowers under the hooves of horses, presumably they were saffron (Aucante, 2000). Exploring several carvings from Sumeria indicates that these clever people traveled from Iran to Mesopotamia about 6000 years ago and established the first saffron farms about 3500 years ago. They wrote 108 lines on a historical tablet regarding the methodology of seedbed preparation and cultivation the crop (Abrishami, 2004; Deo, 2003). Based on some traditional stories during the late Achamenid age (an Iranian dynasty), the agricultural and medicinal documents after being translated to Greek, were destroyed by Alexander. A saffron-based pigment was found in a 50,000-year-old depiction of prehistoric northwestern Iran (Wikipedia, 2019b). A printing from Knossos, Crete (1700 BCE) indicated that the ancient Greek farmers probably worked toward Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00004-6 © 2020 Elsevier Inc. All rights reserved.

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SECTION | II Safron production

FIGURE 4.1 “Young SaffronGatherer,” detail from the “Saffron-Gatherers,” wall painting from Thera, Greece, 1650 BCE [after Christos Doumas, the wall paintings of Thera (Athens: Thera Foundation, 1992)]. From Wikipedia, 2019b. Saffron. Available from: ,https://en.wikipedia.org/wiki/saffron..

domestication of saffron between 3000 and 1600 BCE (Deo, 2003). It is one of the most historical and traditional crops in that its cultivation is based on traditional knowledge and has had little contribution from modern technology (Fig. 4.1). It is a crop with minimum water needs, and under severe restrictions and conditions it provides remarkable income for farmers. Time has been devoted by scientists since ancient times to outline the process of saffron domestication (Theophrastus 371
287 BCE, Pliny CE 23
79, Dioscorides CE 1st century) (Deo, 2003; Negbi, 1999). From the Middle Ages to the industrial revolution, saffron constantly increased in commercial value. Due to high medicinal value and antioxidant ability, it is widely used in human diets (Abrishami, 2004; Grilli Caiola, 2010). In physiological, ecological, and cultural practice aspects, it has considerable differences with other crops. Its flower usually appears before other organs, the onset of flowering corresponds with cold weather of fall, and only a small portion of it flowers (i.e., stigmatic arms and style) are the most valuable part of the plant. Negbi and Negbi (2002) suggest that saffron was first harvested from Crocus cartrightianus, a mutant of which is C. sativus, which is distinguished by having elongated stigmas. It was selected and domesticated in Crete in the late Bronze Age. Furthermore, saffron was established as an expensive crop from east to west (Negbi, 1999; Warburg, 1957), but this historical crop has gained little from modern technology and it is mainly supported by traditional knowledge. It requires a minimum amount of water, and under harsh circumstances it earns relatively satisfactory income for farmers. The glory of saffron has increased since 1980 and draws the attention of several authors (Basker and Negbi, 1983; Greenberg and Lambert Ortiz, 1983; Tammaro, 1990). Research on saffron cultivation is performed in various countries including Iran, Azerbaijan, Italy, Greece, and India. However, the evolution of this triploid plant, which exclusively propagates by corm, is not well understood (Aghayev et al., 2009; Rashed-Mohassel, 2006). Several studies focus on its origin. There is still a ambiguity around whether is it a mutation from wild species by autotriploidy (Mathew, 1999; Negbi and Negbi, 2002), or if it is an allotriploid in which one parent is C. cartrightianus. Therefore more investigations concerning the morphology, phenotype, and life cycle peculiarities of this species as well as molecular analysis seems inevitable (Kerndorff et al., 2015). Furthermore, the most prominent subjects at hand to increase saffron production are ways to increase saffron yield per area (Negbi et al., 1989), development of corm production in vitro, cultivation of the stigma of saffron itself, and improvements via crossing saffron with its close relatives (Chichiricco, 1990; Negbi, 1999). In the following sections attempt has been made to look at the botany of the Crocus genus with special attention to probable crosses between the species that resulted in triploid saffron production. For better understanding of the genus Crocus and its position in the plant kingdom, we start from the iris family and species of saffron in brief. We will pay particular attention to cultivated saffron, which is a strategic crop in Iran and some other countries.

4.2

Crocus

Little attention was paid to the genus of Crocus until the decade 1970 (Basker and Negbi, 1983; Greenberg and Lambert Ortiz, 1983). A comprehensive study of Crocus began in 1983 by Kerndorff et al. (2015) and is still ongoing.

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These researchers are undertaking a systematic investigation of Crocus (Kerndorff et al., 2013) and molecular analysis concerning the relationship among species. In their most recent investigation, they suggest to look at morphological characteristics of this genus in more detail. They also provide detailed insight into the Crocus phylogenetic relationships, primarily based on morphological and phenotypic parameters of Crocus species. More information is needed regarding the habitats of Crocus species as well as life cycle and morphological parameters involved in distribution and diversity of different species (Kerndorff et al., 2015). Species of Crocus are herbaceous and are mostly grown in the Mediterranean region from Morocco and Portugal up to Russia, Iran, and Sin Kiang in western China. They prefer Irano-Turanian areas with cold winters, little rain, and hot summers. They grow actively from fall to midspring and survive in the soil due to their fleshy hard corm covered with coriaceous, membranous, or reticulate tunica. They have beautiful showy flowers and are grown as house plants and ornamentals in home gardens, rock gardens, and parks (Rashed-Mohassel, 2006). They are dispersed in vast groups under optimum environmental conditions. Crocus species are perennials and their life cycle starts with seed germination and various developmental stages in their first year. After 3
5 years it will be a mature plant composed of a solid corm, which is a compressed stem below the ground (Maw, 1886). Adventitious absorbing roots, contractile roots, and absorbing contractile roots are located at basal part of the corm (Kerndorff et al., 2015). Prophyll develops from the base of the pedicle. Bract and bracteole (rudimentary leaves) grow from the base of the ovary. Leaves and flowers are protected within 10
14 cataphylls (modified leaf segments that are broader than true leaves), which develop around the corm and protect the ascending flower buds. Leaves are 5
30 mm wide, 10
18 cm long, and may be navicular or graminaceous type and synanthous or hysteranthous (Mathew and Brighton, 1977). Flowers are solitary or in small groups and are protected within cataphylls. Floral spathe (bract and bracteole) are diphyllus or monophyllus, depending on presence or absence of the bracteole. Flowers are showy with vivid white, yellow, purple, blue, and variegated colors (Rashed-Mohassel, 1989). Different species of Crocus are either fall flowering or spring flowering (Mathew, 1982; Rashed-Mohassel, 2006). Unsurprisingly, the ancestor of saffron is thought to have been fall flowering. Fall flowering species germinate seeds from September to November. In spring flowering species, blooming is from midwinter to spring months and depends on environmental conditions (Kerndorff et al., 2015). Crocus seeds mostly retain the ability to germinate after being dormant for one or more years in the soil. In the first year Crocus develop a corm about 5
8 mm. During hot and/or dry summer, the corms turn dormant and other plant parts die off. Declining temperature results in the dormant corms waking up for a new cycle. Due to low temperature and lack of humidity, for the first season the Crocus has no roots; however, the situation will change after that and the corms will develop roots in the following years. Species of Crocus may prefer high mountain areas or lowlands (Kerndorff et al., 2015). High mountain Crocuses have a corm with long dormancy. After summer nothing of remark is seen of the Crocus above ground until next spring. Lowland Crocuses such as the Mediterranean coast Crocuses have favorable conditions during fall and produce roots, shoots, and new leaves for the next season. However, under favorable conditions when snow melts away and enough humidity for roots is available, in some species, the young Crocus produces two or more leaves for the same season. On the Mediterranean coast, conditions are more favorable during winter and lowland Crocus species produce roots and shoots more often during the normally dormant period. They produce leaves much earlier than low mountain species in the fall and winter. Prolong drier periods in such areas are frequent and this condition forces Crocus to produce leaves by the first spell of heat, which pronouncedly happens earlier than in high mountains and with enough soil moisture for photosynthesis. Therefore we may divide species into four categories based on habitats and Crocus species (Kerndorff et al., 2015): 1. Lowland fall flowering species: These are dormant in summer but active in winter after flowering during fall. They are present around the Mediterranean coast up to 1200 m altitude. They are mostly synanthous, but sometimes leaves may be produced earlier than flowers (hysteranthous) after the hot weather of summer is over. This is more favorable since it results in the new corm ripening faster before the leaves die off in summer. 2. Lowland spring flowering species: These Crocuses are active in winter from February to April or May. Leaves may be produced simultaneously or before flowers. In this group the leaves have to mature in a short period. 3. High mountain fall flowering species: The species of this group produce flowers prior to leaves and, to some extent, the leaves are useless due to snow covering most of winter. However, the flowers are produced periodically before the severe frost. In this situation the flowers wither and the capsule develops underground using energy of the new corm. By spring, the leaves grow quickly and the capsule is pushed upward by the peduncle and the seeds release on the ground. Finally, the leaves fall off and the corms become dormant.

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4. High mountain spring flowering species: These areas are characterized by a short summer when dormancy ends. The corm starts to produce leaves and flowering shoots, but they stay in the ground. With increasing moisture, roots start to grow. As soon as the winter ends, the flowers develop followed by leaf development within weeks or it is possible that the leaves and flowers develop simultaneously. After fertilization the flowers shrivel quickly, the capsule pushes toward the soil surface, and seeds shed early in the summer. Meanwhile, before the corm enters dormant phase, the new corm matures on top of the old one. Based on some observations, saffron (C. sativus L.) has the capacity to grow under either highland or lowland conditions, but flowering postpones under lowland and higher temperature conditions (Molina et al., 2005, 2004).

4.3

Contractile roots

The seeds of Crocus normally germinate on the soil surface. However, the corm is found up to 20 cm deep in the soil (Fig. 4.2A). Therefore some power pushes the corm deep into the soil as explained by Maw (1886). The daughter corm produces on top of the mother corm by using its stored materials. Therefore a self-protective mechanism must send it deeper to the ground. Putz (1992) mentioned the pulling force of contractile root and measured the force for the first time. The contractile root force is observed in several species in Iridaceae and Amaryllidaceae (Fig. 4.2B and C). They are able to move the plants deeper into the soil with a gradual twisting action and contracting their cells. This is a survival mechanism for many geophyte plants of Iridaceae and Amaryllidaceae (Fig. 4.2D). Most likely based on a pulling effect, the bark cells of roots expand longitudinally and act like a rubber band, while turgor pressure acts as a counter pressure to pull it deeper into the ground (Kerndorff et al., 2015) about 1
10 cm each season. When the corm reaches to the optimum depth the contractile root function stops. Contractile roots are temperature, soil texture, and light dependent. The deeper the corm is, the less temperature fluctuation and the less contractile root growth is observed (Molina et al., 2005). Soil textures also affect contractile root formation. Soils with fine texture may not have contractile roots, but soils with coarse texture stimulate contractile root growth (personal observations). Therefore in contrast to absorbing roots, we may not see contractile roots in all Crocus species and all environmental conditions (e.g., in cultivated saffron we rarely see contractile roots unless the soil texture is coarse and sandy). FIGURE 4.2 (A) Germination of Crocus vernus seed, (B and C) contractile root and down-pull, and (D) turn of corm by contractile roots. From Kerndorff, H., Pasche, E., Harpke, D., 2015. The genus Crocus (Liliiflorae, Iridaceae) life cycle, morphology phenotypic characteristics, and taxonomical relevant parameters. Stapfia 103, 27
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After the dormant period corms produce cataphyll within the soil. In the case of young and shallow corm, light may fall on the corm and signal the plant that the corm is shallow; such a signal induces contractile root growth whereas, no contractile roots are produced in the dark. Contractile root growth before its optimum depth is vertical relative to the corm, but in optimum depth, it will be horizontal. Mathew (1982) divided the Crocus genus into subgenera Crocus and Crociris. He then divided the subgenus Crocus in two sections (series), Crocus and Nudicarpus. The common features of the Crocus section are morphology of corm, tunic, bract, bracteole, leaves, flower, seeds, times of flowering, and cytological and ecological features. Other features belonging to the Crocus series are: anthers with extrosedehiscence, scape subtended by membranous prophylls and enclosed and ridden within the sheathing leaves (cataphylls), finely reticulated corm tunic, fall flowering, numerous leaves (5
30), flaccid bract, membranous white or transparent perianth tube, yellow anthers, three branches style expanded at the apex, and papillae seed coat. In the following sections, the focus will be on different aspects of Crocus series with special attention to those species with probable characteristics delivered to saffron (C. sativus L.). However, since the presence of corm is a common feature throughout the genus, we recognize the Crocus corm genus in the broad sense and Crocus corm series in the narrow sense.

4.4

Corm

Crocus corms have different shapes including subglobose (C. angustifolius), subglobose flattened at base (C. michelsonii, C. korolkowii), depressed globose (C. speciosus), disk like flattened (C. gilanicus), depressed globose flattened at base (C. cartwrightianus, C. pallasii, C.thomasii, C. flavus), flattened globose (C. hadriaticus, C. biflorus), depressed ovoid globose (C. sieberi), ovoid (C. almehensis, C. caspius), and elongated-ovoid (C. hermoneus) (Kerndorff et al., 2015; Maw, 1886) (Fig. 4.3). A mature corm is very nutritious and energy rich for the next season after dormancy. Starch (48%), water (39%), sugar (6%), and albuminous compounds (3.2%) are the main content of C. vernus during corm dormancy period in November (Kerndorff et al., 2015). Some predominant undeveloped buds on the corm may develop to cormlets (Maw, 1886). Kerndorff et al. (2015) did not observe a relationship between root production and corm size. However, they observe a positive relationship between production of roots and floriferous power of Crocus. They suggest this relationship may have a roll in the establishment of the phylogeny of Crocus (Harpke et al., 2013). Wolter (1990) mentioned presence of calcium oxalate as a feeding repellent in corm tunica. He isolated and observed the crystals under polarized light. Since, there is not great variation concerning crystals it remains an open question, probably for Crocus phylogeny in the long run. However, he found different orientation, density and size in all Crocuses around the dead vascular bundles of corm tunics. Crysltals are mainly needle like (about 90%), prismatic, or rarely sand like and sometimes combinations of two types. Corm tunics may be considered as some aspects homologous to leaf sheath and some of them may reach up to green needle type leaf. Corm tunics may stay partially outside on the neck when partially located inside (Mathew, 1982). The outside part of the tunic is composed of weak and thin tissue materials located between dead vascular bundles and may be easily decomposed by soil acidity, bacteria, or fungi. The inside part looks more like cork and makes the backbone of the outer part of the tunica. It is more stable and is protected for a year or more until they are pushed outward by new one. The neck is membranous in all kinds of tunica. Tunica may be (1) nonfibrous or (2) fibrous, and the outer part of fibrous shoots may or may not have membranous tissue between them. Fibrous tunica may be interwoven, parallel, reticulate, or a combination of the last two (Kerndorff et al., 2015). Since all species of Crocus series and C. sativus L. possess fibrous tunica, we focus on this series and for nonfibrous tunics refer the reader to Kerndorff et al. (2015). Fibrous tunics: Fibrous tunics are characterized by fibers of different sizes and types of transitions between fibers. They are circular or near circular. The fibers may be parallel, reticulate, a combination of both, or interwoven and may have membranes in between or not. The coriaceous tunics have two different kinds of splits. One includes parallel bands of 0.5 mm width, running from the base upward to the tunic center. The other one, which splits shortly down from the apex, is tooth-like and creates a collar-like neck. Splits in both types never meet. In order to gain a thorough understanding of Crocus and saffron it is better to enhance our knowledge of Iridaceae followed by different species of Crocus, especially those having more similarities with saffron (C. sativus L.).

4.5

Iridaceae

Iridaceae is a family of terrestrial plants, perennial herbs, and rarely annuals with underground stems (rhizomes, corms, or rarely bulbs) from which adventitious roots originate into the soil. Judd et al. (1999) observed large prismatic crystals

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FIGURE 4.3 Corm in different Crocus species. From Maw, G., 1886. A Monograph of Genus Crocus. Dulau and Co., London.

of calcium oxalate are present in the sheath of vascular bundles but not raphide, especially in corms. However, Wolter (1990) found different type of calcium oxalate crystals in corm tunica from which about 90% were needle like of size 7
120 μm. Harpke et al. (2013) established phylogeny of Crocus based on crystal type of species. Tannins and/or terpenoids often are present. Leaves entire, unifacial, alternate, stomatas anemocytic, venation parallel, sheathing at the base, estipulate, edgewise along the stem, or a cluster of leaves basal, elongate, and subulate. Bract and bracteole pronouncedly developed, sometimes leaf-like. Inflorescence determinate, scorpoid scapose, circulates or reduced to a single flower. Flowers are large, trimerous, showy, actinomorph, or zygomorph (Cronquist, 1981; Ghahraman, 1995). Flowers are bisexual and subtended individually by one or two bracts. Iridaceae have six tepals in two rows (3 1 3) that are free or connate at the base or form a flower tube (Mozaffarian, 2001). Iridaceae have three stamens opposite to outer

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perianth, filaments distinct or connate, sometimes adnate to prianths. Anthers may stick to abaxial side of petaloid stigma and style branches. Gynoecium three carpellate and three locular with axile placentation, sometimes monolocular with marginal placentation, ovary inferior. Iridaceae have three stigmatic arms, and stigma and style branches expanded to petaloid with stamens on terminal or abaxial surface of the branches. Ovules are few to plenty in each locule, anatropous, or campylotropous. Fruit a capsule, dehiscent loculicidally; seeds global to oval or pear shape, angular to flat; winged, winglets or arillate, seed coat with cellular structure. Floral formula is as follows: ð4:1Þ Cladistics analysis indicates the monophyly of Iridaceae. However, contradictions exist in phylogenetic analysis. Morphological characteristics relate Iridaceae to Liliales (Stevenson and Laconte, 1995), whereas rbcl sequencing places it within Asparagales (Chase et al., 1995). Recently, attempts have been made to identify plants and their contribution to plant hierarchies based on morphological, serological, and polymerase chain reaction (PCR) analysis. Obviously, the more this clarification shows the historical phylogeny relationship among the plants, the better it will be. Presently, we believe these are 85 genera (Judd et al., 1999; Mathew, 1982) and 1750 species within Iridaceae. The main genera are Gladiolus (250 species), Iris (250 species), Sisyrinchium (100 species), Crocus (83 species), Romulea (90 species), Geissorhiza (80 species), Babiana (65 species) and Hesperantha (65 species). Commonly, Iridaceae is subdivided into the four following tribes (Dahlgreen et al., 1985; Judd et al., 1999): 1. Isophysidoideae with single flowers, superior ovary, and long style branches (e.g., Isophys). 2. Nivenioideae with nectar on petals, separate blue flowers, long style branches, stigmatic branches apically (Judd et al., 1999), some species woody (e.g., Aristen). 3. Iridoideae with nectar glands and long style divided below the anthers and extended into threefolded stigmatic areas (e.g., Sisyrhincheae in which styles are alternate with stamens, Irideae in which stigmatic areas and styles are petaloid, Mariceae and Tigrideae). 4. Ixioideae including Crocus, Gladiolus, Romulea, Syringodea, and Geissorhiza. These are monophyletic based on connate tepals, sessile flowers, operculate pollen with porous sculpturing exine, closed leaf sheath, and having corm (Rashed-Mohassel, 2006). In Ixioideae to which the genus Crocus belongs, similarity is observed between Romulea, Syringodea, and Crocus. Syringodea and Romulea both have ovaries below the ground and actinomorphic and sessile flowers. However, the close relationship between these two genera and Crocus is not quite clear. Convergent evolution likely resulted in some similarities. The leaves of Crocus are dorsiventral and navicular. Species of Crocus might be fall, winter, or spring flowering. The stigma and style of Crocus vary and they may be triparted or digitately dividing, are mostly yellow or red color, and are rich in crocin and saffranal (Duke, 1987; Zargari, 1993). Species of Crocus, especially cultivated saffron, are of medicinal value and some species are used as ornamental and/or are wild. In the following section attempt will be made to cover different species of Crocus sensuslato and cultivated saffron sensu stricto.

4.6

Saffron evolution and phylogeny

Although separation of plant species from each other has been demonstrated by several authors, the evolution of saffron (C. sativus) is an open question (Chichiricco, 1990; Mathew, 1999, 1982; Negbi and Negbi, 2002). We are not aware how saffron originated from wild species. Ambiguity also exists about Iridaceous phylogeny to which saffron belongs to (Goldblatt, 1990; Lewis, 1954; Mathew, 1999). An evolutionary change, which may happen at the levels of populations and species depends on (1) chromosomal changes (mutation), (2) natural selection as guiding factors, and (3) reproductive inoculations (diversification and colonization) (Stebbins, 1976). Presumably every taxon has only one phylogeny and evolution occurs at different rates in different parts of the same organism. Therefore in some plants we may observe both relatively primitive and advanced characteristics due to the constant nature of the processes of evolution. Variability of phenotypes, genotypes, and environmental conditions depends on certain places and times (Bessey, 1915). The numbers of plant species in the world are numerous and explanations for each plant is not easy; however, we may classify the plants based on discontinuity of variations. Discontinuity is apparent in living organism. In other words, anybody is able to recognize wheat from wild mustard.

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Why is this discontinuity observed within the plant world? For instance, Brassicaceae has specific characteristics that we recognize as distinct from Poaceae. Wheat and wild mustard have properties and living criteria that allow us to place them in their specific group. Only the plants with characteristics suitable for specific conditions will have priority to establish and survive in a select environment. In fact, classifying plants based on common characteristics is the only possible system and discontinuities within variation help us to overcome the problem. Why does discontinuity of variation in the plant world occur? In order to survive, each plant in an environment has to adapt those conditions. Obviously, plants with different characteristics being arbitrarily next to each other may not adapt themselves to a select environment. Plants that adapt to those conditions will have priority to survive. Suppose you have different sized rocks in a bag and a mixture of rocks and wood chips in another bag. You are told to divide the contents of each bag. For the first bag, the only way to classify it is based on the size. You put the largest on one side, the smallest on the other side, and in between you arrange them in terms of size. In other words, there will be continuity from large to the small. In the next bag you can separate them in two discrete heaps, one heap containing rocks and the other heap containing woodchips. In living plants and animals, we observe discontinuity of variation. Willow is different than corn and the difference is like wood chips and rocks. Why are combinations of characteristics in plants different? In fact, each plant has its own characteristics and nature for a specific environment. Obviously, if different aspects of plants are put together randomly, they may not tolerate a select environment. In the process of natural selection, those characteristics suited to a specific environment will survive. Where gaps occur and to what degree they occur help us to classify plants. Therefore the plant characteristics we observe today were successful in the process of evolution and the gaps are characteristics not successful enough to evolve or persist (Jeffrey, 1982). The description presented by Judd et al. (1999) likely illustrates production of discontinuity among traits, which selected for a new species (Fig. 4.4). Supposedly, over time mutation may split a population in two, each of which establishes its own ancestor-progeny trait. One (right) produces red flowers and the other (left) produces woody stem. This is evident in evolutionary processes. Red flowers and woody stem are evident of two new populations. This phenomenon repeats within each population (e.g., some woody stem may have fleshy fruits while other possesses spiny seeds). Some red flowers may have four stamens while other may have pubescent leaves (Fig. 4.5). Production of new traits may occur repeatedly and continue through generations resulting in production and diversification of new species. Over time, a new characteristic may become the ancestors called monophyletic (Fig. 4.6). Cladistics analyses of ribulose diphosphate carboxylase of chloroplast revealed the monophyletic group of Iridaceae, which is related to but undoubtedly more advanced than Liliaceae (Chase et al., 1995; Goldblatt, 1990). In contrast to most plants saffron is triploid. Several authors from Iran, Azerbaijan, Pakistan, and Italy investigated and found in saffron 2n 5 3X 5 24 and the basic chromosome number X 5 8 (Aghayev, 2002; Ghaffari, 1986). Although a few scientists believe that saffron is autotriploid derived naturally from C. cartrightianus (Mathew, 1982). Most others believe it is allotriploid. The saffron genome contains evidence of allotriploidy: (1) seven triplets, triplet 5 is a pair and a single chromosome and it is different in morphology, size, and heterochromatin orientation. This triplet indicates that C. sativus is the result of two progenies, one of which donates a single and the other provides a pair of chromosomes. Therefore it is allotriploid (Aghayev et al., 2009). (2) Inter retrotransposon ampliphide polymorphism (IRAP) molecular markers (based on variation in genome) also indicate allotriploidy. (3) Providing apocarotenoid with phylogenetic analysis of the gene Bchi (a key gene involved in biosynthesis of apocarotenoid). Therefore allotriploidy is more conceivable with experimental evidence. However, there are different views about the progenitor of saffron from different authors including:

FIGURE 4.4 Evolution of two hypothetical species. A mutation in progeny (left) produced woody species from herbaceous, similarly a mutation (right) produced plant with red flowers. These characteristics transmitted through generations. x White petals, herbaceous stems; K white petals, woody stems; ’ red petals, herbaceous stems. From Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.E., 1999. Plant Systematics: A Phylogenic Approach. Sinauer Associates Inc., Sunderland, MA.

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FIGURE 4.5 The hypothetical plants of Fig. 4.4 in which two new outcomes resulted in new species. x Petals white, stems herbaceous, leaves nonhairy, stamens five, fruit dry, seed coat smooth Petals white, stems woody, leaves nonhairy, stamens five, fruit dry, seed coat smooth Petals white, stems woody, leaves nonhairy, stamens five, fruit dry, seed coat smooth Petals white, stems woody, leaves nonhairy, stamens five, fruit fleshy, seed coat smooth ’ Petals red, stems herbaceous, leaves nonhairy, stamens five, fruit dry, seed coat smooth Petals red, stems herbaceous, leaves hairy, stamens five, fruit dry, seed coat smooth Petals red, stems herbaceous, leaves nonhairy, stamens four, fruit dry, seed coat smooth From Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.E., 1999. Plant Systematics: A Phylogenic Approach. Sinauer Associates Inc. Sunderland, MA.

FIGURE 4.6 Summary of Fig. 4.5 illustrates the events that resulted in the formation of new characteristics and species. From Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.E., 1999. Plant Systematics: A Phylogenic Approach. Sinauer Associates Inc. Sunderland, MA.

1. 2. 3. 4.

C. cartrightianus and C. hadriaticus (Castillo et al., 2005); C. cartrightianus and C. oreocreticus (Fernandez, 2004); C. cartrightianus and C. thomasii (Mathew, 1999); and C. cartrightianus and C. pallasii subsp. Hausknechtii (Harpke et al., 2013).

The three fertile diploid, 2n 5 16, fall flowering species are more probable to be the putative ancestors of sterile saffron including: 1. Aegean C. cartrightianus; 2. Cretan C. oreocreticus; and 3. Italian C. Thomasii. Crosses have happened in the last one (Chichiricco, 1990) and showed varied and viable numbers of hybrids. Since C. sativus vegetatively propagates, genetic improvement may not occur. However, it may fertilize with both species of C. cartrightianus and C. thomasii. On the other hand, these two species are self-sterile but cross fertilize. Therefore although little is known about the possible ancestor of saffron, the most probable ancestors are C. cartrightianus and C. thomasii, although they have different distribution areas (Brighton, 1977; Mathew, 1982).

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DNA of isolated nuclei and mathematical pairing of DNA confirmed the natural relationship and similarities between C. sativus and C. cartrightianus (Brandizzi and Grilli Caiola, 1998), but DNA was less in C. thomasii. G-C content in C. sativus was homologous in different colons and similar to C. cartrightianus but lower than C. thomasii. However, C. sativus is more similar to C. thomasii but DNA of C. sativus are similar to C. cartrightianus though not too far from C. thomasii. Comparing the chromoplast of Crocus species with saffron indicates each species has different composition, but on the whole the chromoplast of saffron looks more like C. cartrightianus and C. thomasii. The overall pigment composition and amount in C. cartrightianus and C. sativus are similar and lycopene may be observed in the flower tube but not in the stigma. The karyotype of C. sativus is similar to that of C. cartrightianus and C. thomasii. After saffron passed its original evolution, it has been cultivated since 3500
4500 years ago. Because it was grown in different countries with different climates, it likely underwent different stressful factors and different sources of mutation and recombination in spite of sterility (Aghayev, 2002; Rashed-Mohassel, 2006). In conclusion, to study autotriploidy of saffron, C. cartrightianus may be the best species to use, but if one believes on allotriploidy, it is better to work on C. cartrightianus as the first progenitor of saffron and for the second progenitor consider C. thomasii, C. pallasii and the other formerly mentioned species as priorities for study, respectively. It requires a genome wide analysis and crosses of different species of Crocus, which is labor intensive and time consuming. Certainly, with progressing modern technology and instrumentation, the next generation of researchers will develop promising new insights into the phylogeny and biosynthesis of different compositions of saffron and will more decisively answer these questions. Answering these questions will require embracing of multidisciplinary challenges such as paleobotany, systematic, histology, molecular biology, physiology, and related areas. In order to become familiar with the probable progenitor of saffron, descriptions of the pertinent species are introduced in the following section.

4.7

Pollination and seed growth

Pollen is required for pollen germination followed by fertilization and seed formation. Since the Crocus flowers are mostly showy with vivid colors, the pollination is by insects (entomophily). Flowers are hermaphroditic with enough pollen and nectar for insects to land on it. Pollination starts when the flowers open. In most spring flowering Crocus, the flowers open up when the temperature is around 15 C, but for high mountain species, the flowers open earlier when the temperature is between 5 C and 10 C. Opening the flowers under sunny and dry weather conditions resulted in quick ripening of the pollen sac and microspores (Grilli Caiola and Canini, 2010). The pollen sacs open longitudinally by a cohesion mechanism and shed the pollen. Crocus’ flowers are allogamous; the generative and vegetative nuclei transformed from the stigma toward the style band produce the pollen tube with a vegetative nucleus ahead followed by a generative nucleus within the tube nucleus. When the tube nucleus reaches to the ovule micropyle, the vegetative cell gradually disintegrates and the generative cells divide into two sperm. One sperm fertilizes the egg within the ovule and the other one fuses to the secondary nucleus, and therefore a double fertilization occurs. Fertilization of the egg with the first gamete produces the germplasm and fusion of the second gamete with the secondary nucleus produces endosperm (albumen) at the expense of the nucellus within the embryo sac of the ovary. Simultaneously, the ovule integuments develop and turn into seed coat or testa. Therefore the seed consists of embryo or germplasm, endosperm, and seed coat. At the same time, the ovary wall develops into a capsule fruit. This is the normal case of pollination and seed development. C. sativus is triploid and sterile, therefore triploid saffron leads to abnormal chromosome pairing, irregular chromosome distribution, and infertile gametes (Chichiricco, 1987; Rudall et al., 1984). However, rarely we observe sexual reproduction and seed formation.

4.8

Sexual reproduction

Saffron is triploid; therefore abnormal chromosome pairing occurs at prophase and results in infertile gametes. Abnormal microspores produce abnormal pollen found in triploid. Pollen abnormalities are based on shape and dimension. During meiotic division abnormalities found in triploids another word, microspores display cytoplasmic degeneration resulting in an incomplete meiosis and abnormal microspore. The number of abnormal microspores is much lower in C. sativus than C. cartrightianus (Karasawa, 1933). Stigmas have three independent canals each of which extends toward locular ovary. Ovule orientation within ovary, megasporogenesis, and development of the embryo sac occurs as in other iridaceous plants with seven cells within the embryo sac (Chichiricco, 1989a,b, 1987). Incompatibility in pollination exists in saffron and cross fertilization between C. sativus and other species is also limited (Grilli Caiola, 1999; Grilli Caiola et al, 2001). Infertility in pollens is much higher than in ovules (Chichiricco, 1984). In triploids, during meiotic division abnormalities are observed frequently. Irregular chromosome assortment occurs and the production of

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FIGURE 4.7 Cultivated saffron (Crocus sativus L.), Mathew (1982, 1999). Syn. C. sativus var. officinalis L. C. officinalis var. sativus Huds. C. sativus var. cashmirianus Royale C. orsinii Parl C. sativus var. C. orsinii (Parle) Maw (Ro) From Wikipedia, 2019b. Saffron. Available from: ,https://en.wikipedia.org/wiki/saffron..

megaspores will not be the same and will be genetically unbalanced. Frequently the ovule does not reach the fertilized stage due to unsuccessful megaspore development (Grilli Caiola and Canini, 2010). However, according to one report, if we have 20% (Grilli Caiola and Zanler, 2005) or 10% (Karasawa, 1933) normal pollination, of which 4% produce pollen tubes, and all of them are able to fertilize an ovule (which may not be the case), the number of seeds produced will be much less than 1%. Therefore we may consider the corms as the only source of saffron reproduction. The viable seeds of saffron have greater dimensions compared with diploid Crocus species due to its triploidy. The seed produces small corm and a leaf in the first year. In the second year, it produces a larger corm, which has a small elliptical shape with reticulate tunica. In the third year the corm can produce cormlets. In the following sections, the cultivated saffron and the probable species involved in the production of saffron will be explained (Fig. 4.7).

4.9

Cultivated saffron (Crocus sativus L.)

Saffron is a perennial plant about 20
30 cm tall. Corm are spherical and compact, flat at the base, up to 5 cm in diameter and 50 g in weight, and covered with fine reticulate fibrous tunics extended upward about 5 cm (Fig. 4.8) above the neck of the plant (Rechinger, 1975; Wendelbo, 1977). There may be three types of adventitious roots present based on variable conditions (Negbi, 1999): 1. Absorbing roots: originate from the base of planted corm; they are thin, rather long, and fibrous. 2. Contractile root: is thick and short, developing singly at the base of sprouting buds and acts in deepening the newly formed corm. It may also absorb water and nutrients. 3. Contractile absorbing roots: develop on the parent corm near spouting buds and bear the contractile roots (Fig. 4.9). Cataphylls are 5
11 in number, white, nonphotosynthetic, membranous, protecting new leaves. Leaves 8
10, usually synanthous, appear when we apply late irrigation prior to flowering, especially when the weather is colder. Other leaves appear before flowering (hysteranthous) if we apply early irrigation prior to flowering, under mild weather conditions. Leaves are straight and green, glabrous or ciliate, navicular, lanceolate, 1
3 mm wide and may reach to 50 cm length during early spring (or even more under favorable conditions), greenish gray, pubescent. Bract and bracteole that grow from the base of the ovary are unequal, white, and distinctly exposed from scale like leaves (Fig. 4.10). Corm is a compressed stem below the ground and if we remove the tunica we distinctly see the nodes, internodes, and buds (from which cormlets originates) on it (Figs. 4.11 and 4.12). The number of fragrant flowers from each corm is 1
4 depends on the size of the corm. Flowering time is from October to November. Perigone tubes are 4
12 cm long. Perianth segments are 3
5 cm length and 1
2 cm wide, dark lilac or violet to purple, rarely reticulate venation at collar (Pigantti, 1982; Mathew, 1982), obovate or oblanceolate with blunt tip. The flower tube is white to lilac and pubescent at adnation of filaments. Filaments are 7
11 mm and white to brown purple with hairs on surface and margins. Anthers are

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FIGURE 4.8 Spherical and compact corm with fibrous tunics in C. sativus.

FIGURE 4.9 Absorbing and contractile in C. sativus.

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FIGURE 4.10 Parts of saffron flower.

15
20 mm long. Style are orange red in color and bear vivid red broad club shape stigmatic arms, each of which is 25
35 mm long and originates from the upper half of the anther or at least half portion of perianth segments. Ovary inferior is located with three locular and fused carpels adjacent or below the soil surface (Fig. 4.10). The floral formula is as follows: ð4:2Þ Due to triploidy of saffron, (3n 5 24) (Aghayev, 2002; Ghaffari, 1986; Mathew, 1999), it is self-incompatible and male sterile. Therefore despite of production of stamen and carpels, stigma and ovule rarely seed in nature and it is instead reproduced by selected corms. However, capsule length is 1.5 6 1 cm and seed diameter is 4.2
4.6 mm 3 2.6
3.8 mm. It has a long dormant summer and its vegetative activity is from fall to mid spring when it loses its leaves. It survived the summer as underground corms. Corms produce buds that originate new leaves and flowers. Flowering corms contain 8
12 buds and each sprouting bud gives rise to new cormlets. Large corms are able to produce more leaves and flowers. A positive relationship exists between corm size and flower production. The leaves sprout from buds on a short stem and are embedded by whitish bracts. Experiments conducted in Iran and Spain concerning the phenological stages of saffron obtained the following conclusions (Lopez-Corcoles et al., 2015): 1. Corm dormancy: It starts by the middle of the May after withering leaves. The corms go to dormant phase and apparently no morphological or physiological changes are observed. By the end of the dormant period, roots from the base of the corm form the root plate by late September and early October.

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FIGURE 4.11 Axillary buds, nodes, and internodes in saffron corm.

2. Early leaf and flower development: Leaves and flowers emerge while the cataphylls are wrapped around them. It happens during mid-October and November when leaves and flowers are completely emerged and the roots are also growing. 3. Flowering: Flowers may occur synanthous (simultaneously with leaves) or hysteranthous (flowers after leaf emergence), depending on weather conditions, mainly temperature. The leaves and roots keep growing during this phase. 4. Leaf senescence: By spring with increasing temperature and decreasing soil moisture, the mother corm nutrients are fully transferred to daughter corms. When daughter corms fully develop the leaves senesce basipetally and fall off and the new corms are formed. Lopez-Corcoles et al. (2015) divided phenological stages of saffron to four vegetative (sprouting, leaf development, development and replacement of corm, plant senescence) and two reproductive (appearance of flower cataphylls and flowering) stages and subdivided each stage. Ideally these divisions could be determined exclusively and independently for saffron instead of plugging into phenological criteria for other crops. Saffron is propagated vegetatively by daughter corms developing on mother corms. Each corm is surrounded by a dense mat of parallel fiber and survives only one season. Under favorable conditions each corm may produce up to 10 cormlets. A well-developed mother corm may produce 15
20 cormlets after 4 years. Usually corms beyond 2.5 cm in diameter and 10 g in weight are able to produce flowers. Different species of Crocus are distributed from the Mediterranean region to China, but the acreage of cultivated saffron is increasing in countries such as Turkey, Italy, Pakistan, France, and the United States. There is less ambiguity about the origin of saffron from wild saffron in Greece (Rashed-Mohassel, 2006). Based on plant international code, the name of saffron should change as it has published before. However, it is more rational that wild saffron, which is still cultivated in some areas, be considered a new species, and the familiar name with broad spectrum usage be applied to this plant with valuable commercial product. This practical method should not cause

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FIGURE 4.12 Mother corm and new cormlets in saffron corm.

suspicion. We similarly have other cultivated plants which differ considerably from their wild ancestors such as, garlic (Allium sativa), onion (Allium cepa), and some cereal crops as well as wild saffron (C. cartwrightianus Herbert) (Mathew, 1999) (Fig. 4.13).

4.10

Wild saffron (C. cartwrightianus Herbert)

Wild saffron has perennial corms about 10
15 mm (up to 20 mm) in diameter, depressed globose, flattened at base (Fig. 4.14). Corms are covered with delicate fibers, reticulate, and extend toward the apex of the corm to the neck about 2
4 cm (Fig. 4.14). Adventitious roots occur at the base of the corm. Cataphylls are present (3
5) and are white and membranous, protecting new leaves. Leaves (7
12) mainly synanthous, green, and scattered around the stem are about the same size as the flower, 1.5
2.5 mm in width, and are glabrous or pubescent. Wild saffron is fall flowering, producing 1
5 pale lilac or purplish white flowers. Perianth is mostly white with dark reticulate orientation. Perianth tube is stained dark at the base of the segments, sometimes white with no veining, glabrous, throat white or lilac, ciliate, with pronounce spathe. Bract and bracteole are present and unequal, white, and membranous, gradually tapering toward end. Perigone tube is 3
5 cm and sometimes up to 7 cm long. Inner and outer perianth segments are 1.4
3.2 cm length 3 0.7
1.2 cm width, lanceolate to obovate, glabrous or slightly papillae at the base. Filaments are 3
7 mm and yellow. At least half of the petal segments are located under the anther. Style are three flat club shaped red stigmatic arms. Each arm is between 20 and 27 mm long, exceeding the anther or about the same size of anther. Fruit is an elliptical capsule, 1.5
2.5 cm long and 0.6
0.7 cm wide, located on a short peduncle above the ground level at maturity. Seeds are brownish red, irregularly round, 3
4 mm in diameter. Raphe extend along the seed and terminate into sharp

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FIGURE 4.13 Wild saffron (C. cartwrightianus Herbert). Syn. C. sativus Cibth and Smith (Mathew and Brighton, 1977) C. sativus Linn. var. cartrightianus (Herb) Maw From Marks Garden Plants, 2019. Crocus. Available from: ,http://www.marksgardenplants.com/crocus.htm.. FIGURE 4.14 Types of corm in Crocus species. From Maw, G., 1886. A Monograph of Genus Crocus. Dulau and Co., London.

caruncle (,1 mm). Testa have long dense papilla. Flowering time is October to December. 2n 5 16 for this species (Mathew and Brighton, 1977; Rashed-Mohassel, 2006). C. cartrightianus grows wild and cultivated. It grows mostly in rocky hillsides, lawns, pine and calcareous areas at around 1000 m altitude. Wild saffron is mostly distributed in the Athica and Cyclades regions of Greece. The geographical distribution of this species differs from that of cultivated species; however, specialists believe that wild saffron is the ancestor of cultivated saffron (Mathew, 1999; Rashed-Mohassel, 2006). This species has been observed on Greek islands.

4.11

Crocus palasii subsp. Haussknechtii

This species is perennial with corm that are oval to compact globose, 30 mm diameter, flattened at base. Corm tunic are fibrous, perfectly reticulate, extended about 10 cm upward around the Perigone tube (Fig. 4.14). Cataphylls (3
5) are white and membranous. Leaves number 7
17, rarely 5 (Mozaffarian, 2001). It is a fall flowering synanthous or leaves appear shortly after flowering and may remain on the plants until next growing season. Leaves are grayish green, 0.5
2 mm wide, sometimes pubescent at margins or keel (Rashed-Mohassel, 1989; Wendelbo, 1977). Flowers number 1
6 and are fragrant, pale to deep lilac blue, pinkish or purplish. Perianth segment obovate or obtuse, emarginated round or obtuse, may be notched at tip, rarely sharp pointed, 3.2
4.2 cm 3 0.8
1.6 cm. Inner perianth are usually smaller than outer perianth. Veins are usually darker. Perigone are white, lilac, or purple and pubescent, 4
10 cm. Spathe are present. Bracts and bracteole are also present with unequal, tapering ends and are white, membranous, and surrounded by cataphylls, about 4.5
6 cm length (Mathew and Brighton, 1977; Wendelbo, 1977). Filaments are

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FIGURE 4.15 Joe ghasem saffron (C. pallasii subsp. Haussknechtii). C. palasii subsp. Haussknechtii (Boiss and Reut. Ex Maw) B. Mathew Syn. C. sativus var. Haussknechtii Boiss and Reut. Ex Maw C. haussknechtii (Boiss and Reut. Ex Maw) Boiss From Wikipedia, 2019a. Crocus pallasii. Available from: ,https://en.wikipedia.org/wiki/Crocus_pallasii..

3
13 mm, white and glabrous or sparsely haired. Anthers are yellow, 1
2 cm long. Style and stigmatic arms are abruptly broad, vivid red at distal end. The point of dividing style is located in the upper half part of the anther. Stigmatic arms are 3
15 mm long, about the same level as perianth segments, and broad at the free end. Fruit is an elliptical capsule, 1.5
3 cm long and 0.7
1 cm wide and located on a short peduncle up to the ground level or few millimeters higher than soil surface (Mathew and Brighton, 1977). Seeds are irregularly spherical, 3
4 mm in diameter, and red to purplish with a short caruncle. Testa is covered with hairs. This species is diploid with 2n 5 16. Flowering time is October to November. It grows on rocky dry hills at 1300
2300 m altitude. It is mostly found in the western half of Iran, northeastern Iraq, and southern Jordan. It is one of the most similar to cultivated saffron and also resembles C.cancellatus, in which the style is divided to several yellow branches. Historical investigation by Abrishami (2004) indicates this saffron, which is presently called Joe Ghasem, is the descendent of a wild saffron domesticated in the Zagros slopes and cultivated. He mentions that its final product may be edible saffron. The endemic people in that area used to call it Karkam and Karkamice from the 10 to 18th centuries. Its Parsi name was Karkam and Karkimas means zaffran (Fig. 4.15).

4.12

Crocus oreocreticus Burtt

This species is a perennial with ovoid corm that are depressed, globose, and flattened at the base. It has 10
15 mm in diameter, fibrous, tunic, finely reticulated. New corms develop on top of the older one on the bottom of the tube, with the stem remaining below the ground until the seeds ripen (Fig. 4.14). Leaves number 11 6 4 and are glabrous, 0.5
1 mm wide, and subhysteranthous to synanthous. If leaves are absent at anthesis then they develop after flowering. Cataphylls number 3
4 and are membranous. Flowers are single terminal cup shaped with six tepals in two rows of three. Outer segments are slightly larger and lilac to slightly purple with darker veins. A silvery exterior on the three outer petals differs from C. cartrightianus. Perianth tube is 4
5 cm long. White or lilac perianth segments are subequal, oblanceolate, and obtuse. The inner segments are usually slightly smaller, 1.4
3.3 cm long, and 0.4
1.1 cm wide. Stigmatic branches are 13
20 mm and equal to the tip of anthers. Anthers are yellow, filaments are glabrous and 6 6 2 mm long. Anthers are 10
17 mm. Prophyll, bract, and bracteole are present, white and somehow flaccid. A long style is divided into three and is rarely yellow. The plant is pollinated by insects, bees, and moths. It looks closely to C. cartrightianus, and some people do not consider it a new species. Capsule is 1.5 6 0.2 cm long, 0.7 cm wide on a short pedicel just above the ground. Seeds are red and 3
5 mm long with sharp caruncle. This species is 2n 5 16. It is flowering. During October to November it grows in dry Mediterranean region of Crete mountain sides on limestone. It is an

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SECTION | II Safron production

FIGURE 4.16 Crocus oreocreticus Burtt (1948). From Marks Garden Plants, 2019. Crocus. Available from: ,http://www.marksgardenplants.com/ crocus.htm..

endangered species, protected under international law and cannot be picked up. Ask hotels to donate a small area to native gardens for beautiful species to receive good publicity (Fig. 4.16).

4.13

Crocus Thomasii Tenore

This species is a perennial. The corm is depressed globose, flattened at base to narrowly ovoid. The corm is 8
12 mm in diameter, tunic fibrous, slender, finely reticulated and extended about 1 cm above the neck (Fig. 4.14). Cataphylls number 3
5 and are white and membranous. Leaves number 5
10 and are synanthous, equaling the flowers at anthesis but sometimes the top showing are green. Leaves are 0.5
1.5 mm wide, glabrous, or papillae at margins. Flowers number 1
2 (up to 3), are fragrant, not strongly veined but sometimes veined, pale to deep lilac, violet toward the base of the segments, throat pale yellow, and pubescent. The perigonium is elliptic. Prophyll, bract, and bracteole are present, very unequal, white, and membranous with long tapering flaccid tip. Perianth tube is 3
6 (up to 8) cm long. Perianth segments are 2
4.5 cm long, 0.7
1.5 cm wide, ovate, obovate or oblanceolate, acute or obtuse. Filaments are 5
8 mm long, pale yellow, and glabrous or finely pubescent at base. Anthers are 9
13 mm long and yellow. Style are divided below or at the same level with anther or a quarter ways up the anther. Three bright branches each of 0.7
2 cm length expand gradually to apex. Capsule is ellipsoid, 1
1.5 cm long, 0.5
0.7 cm wide and raised on pedicel about 2.5 cm above ground level at maturity. Seed is globose, 2 mm diameter with pointed caruncle. For this species 2n 5 16. Flowering occurs October to November. It is grown as wild and cultivated in stony slopes or thin scrubs of Italy, Croatia, and Serbia (Fig. 4.17).

4.14

Crocus hadriaticus

This species is a perennial with corm that are depressed globose flattened at base and 10
15 mm in diameter. Tunic is fibrous, finely reticulated, extended to a short neck (Fig. 4.14). Cataphylls number 3
4 and are white and membranous. Leaves number 5
9 and are synanthous, gray green, 0.5
1 mm wide and hirsute. Flowers number 1
3, and are fragrant, white, sometimes stained externally brownish yellow or ovoid at the base of tepals. It rarely has a pale lilac appearance. Throat is pale yellow or white, pubescent. Prophyll, bract, and bracteole are present, white, and membranous. Perianth tube is 3
9 cm and white, yellow, or violet. Outer tepals are slightly larger, 2
4 cm long and 0.7
2 cm wide. Filaments are 3
11 mm long, yellow, white, glabrous or sparsely pubescent at base. Anther is 0.7
15 mm and yellow. Stigmatic branches number 10
16 (up to 20) mm and are less than half the length of tepals segments. An ellipsoid capsule is 1.2
2 cm long, 0.5
0.6 cm wide and raised on pedicel about 4.5 cm above the ground level at maturity (Kerndorff, 1988). Seeds are reddish brown and 2
3 mm long, terminated to a pointed caruncle. Testa have dense mat of hairs. This species is 2n 5 16. Flowering occurs September to December. It grows in grassy areas of Mediterranean climate in southern Greece (Fig. 4.18).

Evolution and botany of saffron (Crocus sativus L.) and allied species Chapter | 4

55

FIGURE 4.17 Crocus Thomasii Tenore. From CalPhotos, 2019. Crocus thomasii. Available from: ,https://calphotos.berkeley.edu/cgi/img_query? enlarge 5 0000 1 0000 1 1106 1 0071..

FIGURE 4.18 Crocus hadriaticus. From Marks Garden Plants, 2019. Crocus. Available from: ,http://www.marksgardenplants.com/crocus.htm..

4.15

Conclusion

Saffron is a valuable crop due to its coloring, flavoring, and aroma. It is an exceptional crop with exceptional growth habits. The history of saffron dates back to 50,000 years ago when a prehistoric depiction was found in northwestern Iran. Archaeological and historical records suggest that it has been known from prehellenic and Hellenic times when farmers were probably working toward its domestication. Records also indicate that in the 10th century BCE saffron was cultivated in Derbena, Khorasan, Iran. Using precise temperatures and irrigation regimes makes it possible to advance hysteranthous flowering or increase flowering simultaneously. Soil with coarse textures may stimulate contractile root growth, which deepens the newly formed corm and establishes the plant into the soil. Saffron is self-sterile and allosterile, but rarely it may cross with C. cartrightianus, C. thomasii, and C. pallasii. Biochemical analysis shows it is more similar to C. cartrightianus rather than from C. thomasii. In crosses of saffron with C. cartrightianus, the progeny look more like C. cartrightianus. The Crocus species are either fall flowering or spring flowering. However, the ancestors of triploid saffron are supposedly fall flowering. Most scientists believe it should be allotriploid rather than autotriploid in which C. cartrightianus is one progeny and the next progeny is either C. thomasii or C. pallasii var. Hausknechtii. Integration of multiple biotechnical approaches and bioinformatics tools for analyzing and integrating data to acquire maximum output clear the way for improving various aspects of saffron. Hopefully, in the future by employing more sophisticated instrumentation with high resolution, most of the unknown points concerning different aspects of saffron will be uncovered.

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Acknowledgment My special thanks to Mr. Hossein Binabaji for helping me to provide some of the photos and Ms. Niusha Valaei, my PhD student, for her sincere assistance and cooperation.

References Abrishami, M.H., 2004. Saffron, From Yesterday Till Today, an Encyclopedia of Its Production, Trade and Use, first ed. Amirkabir Publication, Tehran (in Persian). Aghayev, Y.M., 2002. New features in karyotype structure and origin of saffron Crocus. Cytologia. 67, 245
252. Aghayev, Y.M., Fernandez, J.A., Zarif, E., 2009. Clonal section of saffron (Crocus sativus L.): the first optimistic experimental results. Euphytica 169, 81
99. Aucante, P., 2000. Lesafran. Artesud, Arles, France. Basker, D., Negbi, M., 1983. Uses of saffron. Econ. Bot. 37, 228
236. Bessey, C.E., 1915. The phylogenetic taxonomy of flowering plants. Ann. Mo. Bot. Gard. 2, 109
164. Brandizzi, F., Grilli Caiola, M., 1998. Flow cytometry analysis of nuclear DNA in three species of Crocus (Iridaceae). Plant Syst. Evol. 211, 149
154. Brighton, C.A., 1977. Cytology of Crocus sativus and its allies (Iridaceae). Plant Syst. Evol. 211, 149
154. Burtt, R.L., 1948. C. oreocriticus. Phyton. 1, 224
225. CalPhotos, 2019. Crocus thomasii. Available from: ,https://calphotos.berkeley.edu/cgi/img_query?enlarge 5 0000 1 0000 1 1106 1 0071.. Castillo, R., Fernandez, J.A., Gomez
Gomez, L., 2005. Implication of carotenoid biosynthetic genes in apocarotenoids formation during the stigma development of Crocus sativus and its closer relatives. Plant Physiol. 139, 647
689. Chase, M.W., Duvall, M.R., Hills, H.G., Conran, J.G., Cox, A.V., Equiarte, L.E., et al., 1995. Molecular systematic of liliaceae. In: Rudall, P.J., Cribb, P.J., Cutler, D.F., Humphries, C.J. (Eds.), Monocotyledons: Systematics and Evolution. Royal Botanical Gardens, Kew, pp. 109
137. Chichiricco, G., 1984. Karyotype and meiotic behavior of triploid Crocus sativus L. Caryologia 37, 233
239. Chichiricco, G., 1987. Megasporogenesis and development of embryo sac in Crocus sativus L. Caryologia 40, 59
69. Chichiricco, G., 1989a. Fertilization of Crocus sativus L. ovules and development of seeds after stigmatic pollination with Crocus Thomasii (Iridaceae). Gyor. Bot. Ital. 123, 31
37. Chichiricco, G., 1989b. Microsporogenesis and pollen development in Crocus sativus L. Caryologia 42, 237
249. Chichiricco, G., 1990. Sterility and improvement of saffron CrocusIn: Tamaro, F., Marra, L. (Eds.), Lo Zaffrano L’Aquila, Italy, pp. 99
107. Cronquist, A., 1981. Anintegrated System of Classification of Flowering Plants. Colombia University Press, New York. Dahlgreen, R.M.T., Clifford, H.T., Yeo, P.F., 1985. The Families of the Monocotyledons. Springer-Verlag, Berlin. Deo, B., 2003. Growing saffron, the world’s most expensive spice. NZ Instit. Crop Food Res. 20, 1
4. Duke, J.A., 1987. Handbook of Medicinal Herbs. CRC Press, Boca Raton, FL. Fernandez, J.A., 2004. Biology, biotechnology, and biomedicine of saffron. Recent Res. Dev. Plant Sci. 2, 127
159. Fiore, A., Pizzichini, D., Diretto, G., Scossa, F., Spano, L., 2010. Genomic transcriptomic of saffron, new tools to unravel the secret of an attractive spice. In: Husaini, A.M. (Ed.), Saffron. Global Science Books, UK, pp. 25
30. Ghaffari, S.M., 1986. Cytogenetic study of Crocus sativus (Iridaceae). Plant Syst. Evol. 153, 199
204. Ghahraman, A., 1995. second printing Plant Systematic, Cormophytes of Iran, vol. 4. Iran University Press, Tehran (in Persian). Goldblatt, P., 1990. Phylogeny and classification of iridaceae. Ann. Mo. Bot. Gard. 77, 607
627. Greenberg, S., Lambert Ortiz, E., 1983. The Spice of Life. Michael Josef, Rain Bird, London. Grilli Caiola, M., 1999. Reproduction biology of saffron and its allies. In: Negbi, M. (Ed.), Saffron: Crocus sativus L. Harwood Academic Publishers, Amsterdam, pp. 1
18. Grilli Caiola, M., 2010. Seed structure in Crocus sativus crossing 3 Crocus cartrightianus Herb; Crocus thomasii Ten. and Crocus hadriaticus Herb. Plant Syst. Evol. 285, 111
120. Grilli Caiola, M., Canini, A., 2010. Looking for saffron parents. In: Husaini, A.M. (Ed.), Saffron. Global Science Books, UK, pp. 1
14. Grilli Caiola, M., Zanler, R., 2005. Self-incompatibility in different Crocus species and in Hermodactylus tuberosus (Iridaceae). Acta Biol. Craco. Ser. Bot. 4 (Suppl. 1), 57. Grilli Caiola, M., Di So mma, D., Lauretti, P., 2001. Comparative study of pollen and pistil in Crocus sativus L. (Iridaceae) and allied species. Ann. Botan. 1, 73
82. Harpke, D., Meng, S., Rutten, Kerndorff, H., Blattner, F.R., 2013. Phylogeny of Crocus (Iridaceae) based on one chloroplast and two nuclear loci: ancient hybridization and chromosome number evolution. Mol. Phylogen. Evol. 66, 617
627. Jeffrey, C., 1982. An Introduction to Plant Taxonomy. Cambridge University Press, p. 159. Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.E., 1999. Plant Systematics: A Phylogenic Approach. Sinauer Associates Inc, Sunderland, MA. Karasawa, K., 1933. On the triploidy of Crocus sativus L. and its high sterility. Jap. J. Gen. 9, 6
8. Kerndorff, H., 1988. Observations on Crocus (Iridaceae) in Jordan, with special reference to Crocus moabiticus. Herbertia 44, 33
53. Kerndorff, H., Pasche, E., Blattner, F.R., Harpke, D., 2013. A new species of Crocus (Liliiflorae, Iridaceae) from Turkey. Stapfia 99, 141
144.

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Kerndorff, H., Pasche, E., Harpke, D., 2015. The genus Crocus (Liliiflorae, Iridaceae) life cycle, morphology phenotypic characteristics, and taxonomical relevant parameters. Stapfia 103, 27
65. Lewis, F.U., 1954. Some aspects of morphology, phylogeny, and taxonomy of the South African Iridaceae. Ann. S. Afr. Mus. 40, 15
113. Lopez-Corcoles, H., BrasaRamos, A., Montero Garcia, F., Romero Valverde, M., Montero Riqueime, F., 2015. Phenological growth stages of saffron plants (Crocus sativus L.) according to the BBCH scale. Span. J. Agric. Res. 13 (3), 1
6. Marks Garden Plants, 2019. Crocus. Available from: ,http://www.marksgardenplants.com/crocus.htm.. Mathew, B., 1982. The Crocus: A Revision of the Genus Crocus (Iridaceae). Timber Press, Portland. Mathew, B., 1999. Botany, taxonomy, and cytology of Crocus sativus L. and its allies. In: Negbi, M. (Ed.), Saffron: Crocus sativus L. Harwood Academic Publishers, Amsterdam, pp. 19
30. Mathew, B., Brighton, C.A., 1977. Four central Asian species (Liliaceae). Iran. J. Bot. 1 (2), 123
135. Maw, G., 1886. A Monograph of Genus Crocus. Dulau and Co., London. Molina, R.V., Gracia
Luiz, A., Coll, V., Ferrero, C.V.M., Navarro, Y., Guard Iola, J.L., 2004. Flower initiation in the saffron Crocus (Crocus sativus L.), the role of temperature. Acta Hortic. 650, 39
47. Molina, R.V., Valero, M., Navarro, Y., gladiola, J.L., Gracia
Luiz, A., 2005. Temperature effect on flower formation in saffron (Crocus sativus L.). Sci. Hortic. 103, 361
379. Mozaffarian, V., 2001. Plant Classification. Book II, Morphology and Taxonomy. Amirkabir Publication, Tehran (in Persian). Negbi, M., 1999. Saffron cultivation, past, present and future prospects. In: Negbi, M. (Ed.), Saffron: Crocus sativus L. Harwood Academic Publishers, Amsterdam, pp. 1
18. Negbi, M., Negbi, O., 2002. Saffron domestication in bronze age crete. World island in prehistory. In: Waldern, W.H., Ensayat, J.A. (Eds.), World Island in Prehistory. International Insular Investigations. British Archeological Reports. Archeopress, International series 1095. Negbi, M., Dagan, B., Drop, A., Basket, D., 1989. Growth, flowering, vegetative reproduction and dormancy in saffron Crocus (Crocus sativus L.). Israel J. Bot. 38, 95
113. Pigantti, S., 1982. Flora Ditalia. Edagricola Press, Italy. Putz, N., 1992. Measurement of a pulling force of a single contractile root. Can. J. Bot. 70, 1433
1439. Rashed-Mohassel, M.H., 1989. The identification and distribution of saffron genus in Iran. Proceeding of First Saffron Conference. 8
9 November, Eslam Abad, Ghaen, Iran (in Persian). Rashed-Mohassel, M.H., 2006. Saffron botany. In: Kafi, M., Koocheki, A., Rashed-Mohassel, M.H., Nassiri, M. (Eds.), Saffron, Production and Processing. Science Publishers, New Hampshire, USA, pp. 13
38. Rechinger, K., 1975. Flora Iranica Iridaceae, vol. 112. Academische Druck. U. Verganstalt, Granz. Rudall, P.J., Owens, S.J., Kenton, A.Y., 1984. Embryology and breeding system in Crocus (Iridaceae). A study in causes of chromosome variation. Plant Syst. Evol. 1-2, 119
134. Stebbins, G.L., 1976. Variation and Evolution in Plants. Columbia University Press, New York. Stevenson, D.W., Laconte, H., 1995. Cladistic analysis of monocon families. In: Rudall, P.J., Cribb, P.J., Cutler, D.F., Humphries, C.J. (Eds.), Monocotyledons: Systematics and Evolution. Royal Botanical Gardens, Kew, pp. 543
578. Tammaro, F., 1990. Crocus sativus L. cv Navelli L’Aquila. Saffron: environment, cultivation, morphometric characteristic, active principle, uses. In: F. Tammaro, L. Marra (Eds.), Lo Zafferano: Proceedings of the International Conference on Saffron (Crocus sativus L.) L’Aquila (Italy), 27
29 October 1989, Universita` Degli Studi L’Aquila e Accademia Italiana della Cucina, L’Aquila. Warburg, E.F., 1957. Crocuses. Endeavor 16, 209
216. Wendelbo, P., 1977. Tulips and Irises of Iran and Their Relatives. Botanical Institute of Iran, Botanical Garden, Tehran. Wikipedia, 2019a. Crocus pallasii. Available from: ,https://en.wikipedia.org/wiki/Crocus_pallasii.. Wikipedia, 2019b. Saffron. Available from: ,https://en.wikipedia.org/wiki/saffron.. Wolter, M., 1990. Calciumoxalat-Kristalle in den knollen-Hullen von Crocus L. (Iridaceae) und ihresystematische Bedeutung. Bot. Jahrb. Syst. 112, 99
114. Zargari, A., 1993. Medicinal Plants., vol. 4. University of Tehran Press, Tehran (in Persian).

Further reading Azizbekova, N.Sh, Milyaeva, E.L., Lobora, N.V., Chailakhyan, M.Kh, 1978. The effects of gibberellin and kinetin in formation of flower organs in saffron Crocus. Sov. Plant Physiol. 25, 471
476. Negbi, M., 1990. Physiological research on the saffron Crocus (Crocus sativus L.). In: F. Tammaro and L. Marra (Eds.), Lo Zafferano: Proceedings of the International Conference on Saffron (Crocus sativus L.) L’Aquila (Italy), 27
29 October 1989, Universita` Degli Studi L’Aquila e Accademia Italiana della Cucina, L’Aquila, pp. 183
207.

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Chapter 5

Soil conditions for sustainable saffron production Hamid Shahandeh Texas A&M University, College Station, TX, United States

Chapter Outline 5.1 5.2 5.3 5.4

Introduction Soil conditions Soil texture Soil nutrient content

5.1

59 60 60 61

5.5 Saffron nitrogen requirement 5.6 Nitrogen use efficiency in saffron 5.7 Conclusion References

62 64 65 65

Introduction

One of the primary functions of soil is to serve as a medium for plant growth (Russell, 1912), and plant growth is impacted by soil conditions that influence the availability of water and nutrients and gaseous exchange (Passioura, 2002). However, the soil of saffron (Crocus sativus L.) is not as merely a supportive medium for plant growth but is more of a managerial concept. Saffron is a high-value perennial plant grown for its flower’s stigma this is used as a spice, with stigmas being traded for more than 1500$ kg21 wholesale and for many times more on the world’s retail markets (Golden Saffron, 2017). Saffron soil is usually prepared as a flower bed with tons of manure, compost, and layers of mulch for higher production (McGimpsey et al., 1997; White Book Saffron in Europe, 2006). Saffron as a cash crop is grown in many places around the world from New Zealand to New England (Vermont) and in both greenhouses and fields (McGimpsey et al., 1997; Skinner et al., 2017). In 2005 the five largest producers of saffron in the world were Iran (93.7%), Greece (2.3%), India (2.3%), Morocco (  1%), and Spain (,1%), with saffron being produced in soils in these countries with different soil characteristics and soil orders including aridisols, entisols, inceptisols, vertisols, and alfisols (Gresta et al., 2008; Kirmani et al., 2014; Kumar et al., 2009). Iran currently produces 97% of the world’s saffron (  300 tons) from more than 70,000 ha of mostly calcareous aridisols under irrigation with an average yield of 4
5 kg ha21 (Iran’s Saffron Production, 2016). Saffron grown in greenhouses or fields in Vermont or New Zealand, however, has produced up to four to six times more than average saffron yield in Iran (McGimpsey et al., 1997; Skinner et al., 2017). This difference in yield is partly related to soil management and how the soil growing bed has been prepared. The following is an example of soil preparation for saffron production in Clyde, New Zealand with very high yield (  30 kg ha21) (McGimpsey et al., 1997). Soil raised beds were prepared by incorporating a 2 cm layer of fine (,4.75 mm) coal dust/untreated sawdust mix (50:50 by volume), a 5 cm layer of untreated sawdust, and 0.2 kg m22 (2000 kg ha21) meat and bone meal (an excellent source of N, Ca, P, and some other minerals, K, Mg, Na, etc.) to a depth of 20 cm into gravelly loamy silt soil. A compound fertilizer (N:P:K:S, 12:5:14:4) was also applied in the spring each year at 30 g m22 (300 kg ha21). Saffron corms were then planted at a density of 50 corms m22 (total weight of 1.45 kg corms m22 or  29 g corm) and a 2 cm layer of untreated sawdust was applied as a mulch after planting and topped up during the dormant period each year. Saffron yield in the first year was ,0.5 g m22 (,5 kg ha21), but in the following four seasons the yield mean was 3.73 g m22, or 37.3 kg ha21. In this experiment, soil organic carbon (OC) increased from 2% in 1990 to 5.7% in 1992. The high yield was mainly attributed to the addition of organic matter, sawdust mulch, and corm size. Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00005-8 © 2020 Elsevier Inc. All rights reserved.

59

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SECTION | II Safron Production

In this chapter, soil conditions needed for sustainable saffron production in Iran will be discussed where OC in soils used for saffron production is usually ,1% and the average corm size planted is about 10 g.

5.2

Soil conditions

Agrologically, ecophysiologically, and phenologically, saffron is different from most agronomic crops (Kafi et al., 2006). Saffron grows from corm, a nutrient reserve organ that supports all flower nutrient requirements in the first year of production (Lo´pez Rodrı´guez, 1989). Saffron also has an unusual reverse biological cycle, with flowering occurring in October to November, then vegetative development until May. This reverse cycle is in contrast to most agronomic crops in that the vegetative development is not highly important for production of the economic yield (stigma) but is very important for the production of new daughter corms. In other words, the corms act as the primary nutrient reserve for stigma production and not the soil. Therefore soil conditions for saffron production should be evaluated in the context of corm establishment, growth, and propagation. The most important soil conditions for successful saffron production are high nutrient content, low bulk density (BD), well-developed friable structure, and well-drained but adequate water-holding capacity (Kumar et al., 2009). Among these conditions, soil nutrient content has received the most attention since it can be changed more readily than the others after corms are planted. However, soil texture among all soil properties evaluated has been suggested to be the most significant factor for saffron production in Iran (Ranjbar et al., 2016; Rezvani-Moghaddam et al., 2015). This is because other soil characteristics, such as BD, aeration, infiltration, and drainage that are harder to change, are related to soil texture and can influence all these properties.

5.3

Soil texture

Many studies have shown that saffron can be successfully grown on a wide range of soil textures as long as soils are well drained, with soils being described as calcareous clays (Skrubis, 1990), sandy/loamy (Ferna´ndez, 2004), clays (Sampathu et al., 1984), or sandy soils (Azizbekova and Milyaeva, 1999). Gresta et al. (2008) reviewed the saffron growth on a wide range of soils in major saffron-producing countries. In Spain, the best performance was achieved on well-drained calcareous clay soil, while in Italy the highest yields were obtained on well-drained clay soil or on alluvial deposits with uniform sandy clay or silty texture. In Jammu and Kashmir, India, the predominant soil texture for saffron production was on heavy textured clay loam in the upper horizons and silty clay in the lower horizons. In these countries, the main reason farmers selected these soils was good soil drainage and water-holding capacity was not a limiting factor. But there are a number of other research reports that suggest that mostly sandier soils are used to grow saffron. For example, greenhouse experiments in Iran showed saffron stigma yield in sandy loam soil was higher than in loam and clay loam soils by 39% and 49%, respectively (Khorramdel et al., 2014) or sandy textured soil produced more and bigger corms and higher flower and stigma yields than an original clayey soil (sand was added by 70% to a clayey textured soil to make it a sandier texture) (Rezvani-Moghaddam et al., 2015). Sandy soil has also been shown to affect saffron flower phenology: flowering started on the same day for all soil texture treatments tested, but in sandy soil, flowering ended 3
5 days later and produced the greatest number of flowers and the highest total stigma yield (Gresta et al., 2010). However, these findings cannot be generalized even within the sand soil textural class. For example, in Morocco, soils of different textures were evaluated to determine if saffron might be a sustainable substitute crop with high added value for farmers’ socioeconomical development (Lage and Cantrell, 2009) (Fig. 5.1). This experiment showed that the sandiest soils, S5 and S2, produced the highest and the lowest yields, respectively. In fact, the soil at the site S2 with 72% sand and 3.5% organic matter (OM) produced only 25% of the yield at the S5 site with 82% sand and 2.8% OM. The low yield at the S2 sandy site was comparable to the yield of the clayey soil at the S11 site (Fig. 5.1). Similar results were also observed in diverse soils in farmer fields across a major saffron-producing region in Khorasan, Iran (Rezvani-Moghaddam et al., 2015). Saffron yield of this experiment was related to soil texture, and the researcher reported that the highest yields were achieved in sandy soils and the lowest in clay soils. However, when soils in this experiment were grouped in order of decreasing clay content, the relationship between soil textural class and saffron yield became less significant (Fig. 5.2). Increasing clay content is shown to have a dominant influence on soil water supply, nutrient uptake, and saffron quality (aromatic strength, or safranal content) (Husaini et al., 2010). Unfortunately, in the saffron experiment on Iranian soils, OM content was not reported (Rezvani-Moghaddam et al., 2015). Reported soil OM content of saffron-producing regions in Iran is generally less than 1% (Kafi et al., 2006). Soil organic matter content by influencing soil BD, aeration, infiltration, and drainage could be an important modifying

Soil conditions for sustainable saffron production Chapter | 5

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FIGURE 5.1 Saffron stigma yield at different sites in Morocco for a 2-year production cycle. From Lage, M., Cantrell, C.L., 2009. Quantification of saffron (Crocus sativus L.) metabolites crocins, picrocrocin and safranal for quality determination of the spice grown under different environmental Moroccan conditions. Sci. Hortic. 121, 366
373.

FIGURE 5.2 Effect of soil textural class (C, clay; Si, silt; S, sand; L, loam) on saffron yield. Soil samples were collected from saffron farms in Khorasan, Iran. From RezvaniMoghaddam, P., Khorramdel, S., Mollafilabi, A., 2015. Evaluation of soil physical and chemical characteristics impacts on morphological criteria and yield of saffron (Crocus sativus L.). J. Saffron Res. 3, 188
203 (in Persian).

factor in the degree of saffron response to soil textural change associated with soil aeration and root penetration. For example, root penetration is inhibited in clayey soil at a BD of 1.4 and in sandy soil at a BD of 1.6 g cm23 (Dexter, 2004). Soil OM content can also affect the nutrient content of saffron-growing soils. For example, Fig. 5.1 shows that soils with 2.8%
4.5% OM can produce saffron yields of more than 10 kg ha21 without any fertilizer application in the dry climate of Morocco. A soil with 1% OM could potentially mineralize more than 10
20 kg N ha21 in each growing season (Havlin et al., 2014).

5.4

Soil nutrient content

In most saffron-producing countries, no specific fertilizer recommendations exist for this crop, due to soil organic matter differences, climatic variations, and specific features of the preceding crop and cultivation (White Book Saffron in Europe, 2006). Some researchers even argue that in fertile soils, fertilization is not necessary for successful saffron production because saffron has sufficient nutrients stored in its corms. Considering the small amounts of nutrients removed by saffron, this may not be a farfetched assumption (Table 5.1). Table 5.1 shows the amounts of N, P, and K removed by an average saffron crop in comparison to that removed by an average irrigated wheat crop in Khorasan, Iran (FAO, 2005). To construct Table 5.1 average concentrations of N, P, and K in stigmas, leaves, flowers, and corms were taken from published literature (Aslami et al., 2009; Behnia et al., 1999; Chaji et al., 2013; Katawazy, 2013; Koocheki and Seyyedi, 2015). From this information, it was estimated that for 1 kg of saffron stigmas, flowers or leaves, about 12 g N, 3 g P, and 22 g of K will be removed from the soil

62

SECTION | II Safron Production

TABLE 5.1 Average amount of nutrients removed from a soil by saffron compared to wheat in saffron-producing areas of Iran. Plant part

Nutrients removed N

P

K

N

P

21a

(g kg b

K 21

)

(kg ha )

Saffron stigma

12

3

22

0.06

0.02

0.11

Saffron flower

12

3

22

0.60

0.20

1.10

Saffron leaves

12

3

22

12.0

3.0

22.0

Saffron corm

12

3

22

60.0

15.0

110.0

72.7

18.2

132.2

Wheat grain

21

3

4

63.0

9.0

12.0

Wheat straw

15

1.8

42

45.0

5.4

126.0

108.0

14.4

138.0

c

Saffron total

c

Wheat total a

Nutrients removed with 1 kg of saffron or 1 kg of wheat yield. Average concentrations from available data for all saffron plant parts (Aslami et al., 2009; Behnia et al., 1999; Chaji et al., 2013; Katawazy, 2013; Koocheki and Seyyedi, 2015; Srivastava et al., 2010). c Based on 5 kg stigma (4-year average) and 500 kg fresh saffron flower (50 kg dry), 1000 kg leaves, and 5000 kg corm ha21 (10 g corm, at 50 corm m22) and 2500 kg wheat grain and straw yield. b

(FAO, 2005) and a similar amount of these elements will be stored in 1 kg of saffron corms (Chaji et al., 2013; Koocheki and Seyyedi, 2015). It was reported that at the end of a growing season, a considerable amount of withered dry leaves, or about 1300 kg ha21 (twice the amount of fresh flowers harvested), was left in the field and was not removed from saffron farms in Iran (Behnia et al., 1999). Therefore the only nutrients removed from the soil by saffron production are nutrients taken up by flowers and stigmas, while nutrients removed by saffron leaves are mostly returned to the soil. Nutrients stored in corms are used for the development of daughter plants. Nutrient removal is a crucial element of sustainability and the data in Table 5.1 shows saffron may be one of the closest crops to natural systems sustainability of nutrients. For example, Table 5.1 shows that for an average yield of 5 kg stigma ha21, the amount of N removed from the soil is only 0.66 kg (flower 1 stigma) and the amount of N returned (leaves) or stored and recycled (corm) to the soil is about 72 kg ha21 (leaves 1 corm). The amount of a nutrient removed is sometimes used by depletion models to calculate the amount of that nutrient required for agronomic crops. For a determined yield goal in the absence of established nutrient requirements, this model might be used (Ryan et al., 2009). For example, the relationship N rate 5 Yield goal (kg ha21) 3 0.033 (33 kg N 1000 kg grain21) has been developed for wheat grain yield in a dry climate and suggests that 33 kg N is required for every 1000 kg of yield. Data in Table 5.1 validate this relationship where the recommended N rate was similar to N removed by 3000 kg ha21 wheat yield in Khorasan (FAO, 2005). The question then becomes whether this type of nutrient relationship can be developed and applied to saffron production. However, since the saffron economic yield component (stigmas) generally depends on corm nutrient reserve and not directly on the soil, it may be difficult to develop or interpret a similar type of relationship for crops like saffron. Therefore nutrient requirements for saffron assessed by any method, such as yield goals, mathematical response curves, or balance sheets, should not only be related to stigma yield but also to corm yield. In addition, experiments should be conducted for at least a 3- to 4-year management cycle to determine corm or stigma yield response to nutrient addition. For example, in a 4-year management cycle, the second year usually produces larger progeny corms, which may affect nutrient recommendations the following year (Douglas et al., 2014). All these factors make predicting saffron nutrient requirements a difficult task.

5.5

Saffron nitrogen requirement

From earlier research in Iran in the 1980s it was concluded that N fertilization from either organic (cattle manure) or inorganic sources had the most impact on saffron flowering rates and vegetation and stigma yield (Fig. 5.3) (Kafi et al., 2006).

Soil conditions for sustainable saffron production Chapter | 5

63

FIGURE 5.3 The effects of different combination of N, P, K, and cow manure on saffron stigma yield in an 8year management cycle at two sites located in Khorasan, Iran. Data from Kafi, M., Koocheki, A., RashedMohassel, M.H., Nassiri, M., 2006. Saffron, Production and Processing. Science Publishers, New Hampshire, USA.

FIGURE 5.4 Effect of N and composted cow manure on saffron stigma yield in 3 years (Y1, Y2, Y3) in two sites (Birjand, Ghaen) in Khorasan, Iran. From Behnia, M.R., Estilai, A., Ehdaie, B., 1999. Application of fertilizers for increased saffron yield. J. Agron. Crop Sci. 182, 9
15.

Manure also appeared to be important for promotion of saffron production in soils that were poor in soil OC. In the years that followed, numerous nutrient studies were performed on both short- (1- to 2-year studies for master and PhD students mostly under controlled conditions) and long-term (3- to 4-year field management cycles) bases with different N rates and forms in a number of locations across the saffron-growing state of Khorasan (Aslami et al., 2009). For example, Fig. 5.4 shows the effect of N rates of 0, 50, and 100 kg ha21 from urea and composed cattle manure (0.25% N) at rates of 0, 20, and 40 tons ha21 on saffron yield over 3 years in two major growing sites in Khorasan. Nitrogen fertilizers were applied every year in these locations. The results showed that N fertilization had either a negative or nonsignificant effect on fresh flower weight or saffron stigma yield, and mathematical models, either linear or quadratic, could not explain the saffron response to N fertilizer at either site. However, lower yield at the Ghaen site was related to smaller saffron corm size (Behnia et al., 1999). However, in spite of the inconsistency of saffron response to fertilization and the difficulty in establishing nutrient recommendation rates, growers and researchers rely on past experience or on soil test results developed for other agronomic crops. For example, N is recommended at rates of 50 or 100 kg ha21 year21 from chemical fertilizer and at rates of 30
60 tons ha21 of cattle manure for the first year of saffron production. Composted cattle manure at a rate of

64

SECTION | II Safron Production

60 tons ha21 is among the suggested recommendations for sustainable saffron production (Dobermann and Cassman, 2004) before planting and 20 tons ha21 after the first year (Ghorbani and Koocheki, 2017) or when irrigating with saline water (Sepaskhah and Yarami, 2009). Application of manure to saffron or other crops generally aims to support two major goals: (1) increase the supply of nutrients to the crop and (2) increase the organic matter content of the soil resulting in more favorable soil physical and chemical conditions. These improved characteristics include increasing soil cation exchange capacity; improving soil aggregate stability, soil macrostructure, infiltration, and water-holding capacity; and reducing soil BD and sorption capacity or negative interactions of nutrients with mineral soil (Ketterings et al., 2005). Saffron will generally benefit from manure addition, but there are limitations to the use of manure in saffron-producing areas of Iran, including cost of production and manure availability. Cost is influenced by a dry climate, feed availability for livestock, and labor associated with the production or collection, transportation, storage, and application of manure. Animal manures can be cost effective if the manure is produced on-farm near the site of saffron production. Moreover, the composition of manures can vary significantly depending largely on the animals’ diet, type of animals, and in the ways the manure is collected, stored, and applied. Saffron farmers need much larger amounts of animal manure to supply the same amount of nutrients as from chemical fertilizers, as their elemental concentration is lower than that of mineral fertilizers and the rate of nutrient release from animal manure is also slower. The organic material must be decomposed to release the nutrient elements, which might potentially lead to a nutrient deficient period for saffron. In some literature, it appears there is an assumption that the total N of manure can be readily plant available. In Fig. 5.4, 20 and 40 tons of composted cattle manure with 0.25% N was used with the assumption that it would supply about N equivalent to 50 and 100 kg N ha21 from urea. The nitrogen content of manure is a combination of organic N and inorganic N (NH41
N). Inorganic ammonium is readily plant available (  10%) and the rest (organic N) needs to be mineralized over time (  25% in the first year) (Ketterings et al., 2005). The rate of N release from manure varies with soil texture or cation exchange capacity (CEC), soil pH, soil microbial population, the prevailing temperature and moisture, as well as with soil disturbance by tillage. Moreover, in recommending N fertilizer, upcoming weather and organic matter content of surface soils should also be considered. In general, the nutrients in manure will not be released in one season, with about one-fourth to one-third of N, one-fifth to one-fourth of P, and 1-second to two-thirds of K being released in the first season. An average content of Nitrogen, Phosphorus, Potassium (NPK) in composted cattle manure applied to saffron soils in the Khorasan area is around 1.1%, 0.6%, and 1.2% (on a dry weight basis) (Rezvani-Moghaddam et al., 2015). An application of 30 tons of dry manure with 1% of NPK has been shown to supply the following amounts of nutrients per hectare: 35
82 kg N, 7
21 kg P, and 32
163 kg K in the first year (Bayu et al., 2005). The remaining N in composted manure would behave like soil organic matter N after the first year of application, with declining amounts of N mineralized with time (Meek et al., 1982). Most soils in the Khorasan saffron-growing areas have low levels of soil organic matter, usually between 0.5% and 1% OM, and the application of 30 tons of manure for 2 years could increase soil organic matter content by 2% (Meek et al., 1982). However, there is a dilemma in the use of manure for small saffron farmers in the dry climate of the Khorasan area. Providing 30
60 tons manure ha21 for 70,000 ha of saffron may not be economical or practical, and most saffron farmers will probably use urea since it has been shown to produce similar results as manure (Figs. 5.3 and 5.4).

5.6

Nitrogen use efficiency in saffron

The amount of fertilizer N applied to saffron should be determined not only by profit, but also by potential environmental effects. The efficiency of N applied can be assessed by the partial factor of productivity (PFP) (yield of harvested portion per amount of nutrient applied) and the partial nutrient balance (PNB) (nutrient content of harvested portion per amount of nutrient applied). These nitrogen use efficiency (NUE) terms are easily measured and can identify inefficiencies in order to improve N fertilizer management in saffron. The PFP is intended to answer the question: “How productive is this cropping system in comparison to its nutrient input?” The PNB answers how much nutrient is being taken out of the system in relation to how much is applied. PFP helps explain the return in yield from the use of fertilizer, while PNB tells something of the source of the nutrients removed in the crop. For example, the mean PFP and PNB for wheat in Iran is about 30 kg grain kg21 N and 0.5 kg N removed kg21 N applied (Koocheki and Seyyedi, 2015), respectively, which means wheat farming in Iran has relatively low PFP and PNB, with significantly more N added into the system than recovered. A similar observation has been reported for saffron corm N use efficiency, which showed that the PFP and PNB were 24 and 0.44 with manure at 25 tons manure ha21 and 17 and 0.34 for urea at 100 kg N ha21, respectively. Therefore the NUE terms PFP and PNB for saffron corms are about half of that for wheat production in Iran. Low NUE indicated that saffron did not absorb or utilize N in time or N loss exceeded the rate of plant uptake or increased soil N supply.

Soil conditions for sustainable saffron production Chapter | 5

5.7

65

Conclusion

Saffron, because of its biological and agronomic traits, has been introduced as an alternative crop for low-input agriculture, since it is able to offer good production in sustainable agricultural systems in marginal lands (Gresta et al., 2008). However, chemical fertilizer and weed control treatments and irrigation and chemical are now being used in a systematic manner in all aspects of saffron production in Iran. Saffron is becoming a more attractive cash crop, with chemical fertilizer often being viewed as a panacea for achieving higher yield. The extensive use of chemical fertilizer for saffron production, however, generally has a weak scientific base. Researchers appear to be borrowing nutrient recommendations from common agronomic crops to apply to saffron. Saffron production with chemical fertilizer is now being encouraged beyond its traditional and natural boundaries in Khorasan to more fertile soils of the northern and western parts of the country. Saffron is a crop for marginal lands, where growing other crops is difficult. Saffron is known from earlier days for its low nutrient input, with farmers often adding only meager quantities of organics, such as leaf litter or animal manure, and with no or minimal irrigation for its production. Soil-quality assessment in Khorasan suggests several limitations to the growth of many crops except saffron (Ranjbar et al., 2016). Soil quality may be defined as how well soil does what farmers want it to do (Karlen et al., 1997). Saffron farmers can maintain, manage, and sustain saffron production in the Southern Khorasan ecosystem by the nutrient-buffering power of manure and the use of larger corms. Manure and larger size corms are concepts that should be emphasized and not increasing amounts of chemical fertilizers. Saffron farmers should view themselves more as gardeners rather than farmers and should prepare their soil as they would a flower bed for harvesting flowers. Other saffron-growing countries often argue that they cannot compete in the world market with the saffron produced with low-cost manual labor-intensive farming systems like those in Khorasan, Iran. Technical advances in the future that reduce production costs and expand the saffron production area through more intensive inputs or to move to more fertile soils with better soil conditions may not benefit small saffron farmers in Southern Khorasan. To be able to compete in the future, saffron farmers should be focused on producing saffron organically (using manure buffer power) in Khorasan and taking advantage of their unique climate and environment of optimum temperature that enables saffron production with fewer fungal infections, insects, and weeds.

References Aslami, M.H., Nacuray, E.O., Qaraeen, A., 2009. Saffron manual for Afghanistan, DACAAR, 14
19. Available from: ,https://afghanag.ucdavis.edu/ grain-field-crops/files/saffron-manual.pdf.. Azizbekova, N.S.H., Milyaeva, E.L., 1999. Saffron in cultivation in Azerbaijan. In: Negbi, M. (Ed.), Saffron: Crocus sativus L. Harwood Academic Publishers, Australia, pp. 63
71. Bayu, W., Rethman, N.F.G., Hammes, P.S., 2005. The role of animal manure in sustainable soil fertility management in sub-Saharan Africa: a review. J. Sustain. Agric. 25, 113
136. Behnia, M.R., Estilai, A., Ehdaie, B., 1999. Application of fertilizers for increased saffron yield. J. Agron. Crop Sci. 182, 9
15. Chaji, N., Khorasani, R., Astaraei, A., Lakzian, A., 2013. Effect of phosphorous and nitrogen on vegetative growth and production of daughter corms of saffron. J. Saffron Res. 1, 1
12. Dexter, A.R., 2004. Soil physical quality Part I. Theory, effects of soil texture, density, and organic matter, and effects on root growth. Geoderma 120, 201
214. Dobermann, A., Cassman, K.G., 2004. Environmental dimensions of fertilizer nitrogen: what can be done to increase nitrogen use efficiency and ensure global food security? In: Mosier, A.R., Syers, J.K., Freney, J.R. (Eds.), Agriculture and the Nitrogen Cycle: Assessing the Impacts of Fertilizer Use on Food Production and the Environment, 65. SCOPE, Paris, pp. 261
278. Douglas, M.H., Smallfield, B.M., Wallace, A.R., McGimpsey, J.A., 2014. Saffron (Crocus sativus L.): the effect of mother corm size on progeny multiplication, flower and stigma production. Sci. Hortic. 166, 50
58. FAO, 2005. Fertilizer Use by Crop in the Islamic Republic of Iran. First Version. FAO, Rome, Italy. Ferna´ndez, J.A., 2004. Biology, biotechnology and biomedicine of saffron. Recent Res. Dev. Plant Sci. 2, 127
159. Ghorbani, R., Koocheki, A.R., 2017. Sustainable cultivation of saffron in Iran. In: Lichtfouse, E. (Ed.), Sustainable Agriculture Reviews. Springer, Cham, pp. 169
203. Available from: http://doi.org/10.1007/978-3-319-58679-3-6. Golden Saffron, 2017. Golden saffron, finest premium Persian all red saffron, Grade A 1 , ($8.48/gram). Available from: ,https://www.amazon.com/ Golden-Saffron-Premium-Persian-Highest/dp/B072K71972?ref_ 5 bl_dp_s_web_15526256011&th 5 1.. Gresta, F., Lombardo, G.M., Siracusa, L., Ruberto, G., 2008. Saffron, an alternative crop for sustainable agricultural systems: a review. Agron. Sustain. Dev. 28, 95
112. Gresta, F., Lombardo, G.M., Avola, G., 2010. Saffron stigmas production as affected by soil texture. Acta Hort. 850, 149
152.

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Havlin, J.L., Tisdale, S.L., Nelson, W.L., Beaton, J.D., 2014. Soil Fertility and Fertilizers, eighth ed. Prentice Hall Publishing, Upper Saddle River, NJ. Husaini, A.M., Hassan, B., Ghani, M.Y., Teixeira da Silva, J.A., Kirmani, N.A., 2010. Saffron (Crocus sativus L. Kashmirianus) cultivation in Kashmir: practices and problems. Funct. Plant Sci. Biotechnol. 4, 108
115. Iran’s Saffron Production, 2016. The Iran Project. Financial Times, London. Kafi, M., Koocheki, A., Rashed-Mohassel, M.H., Nassiri, M., 2006. Saffron, Production and Processing. Science Publishers, New Hampshire, USA. Karlen, D.L., Mausbach, M.J., Doran, J.W., Cline, R.G., Harris, R.F., Schuman, G.E., 1997. Soil quality: a concept, definition, and framework for evaluation. Soil Sci. Soc. Am. J. 61, 4
10. Katawazy, A.S., 2013. A Comprehensive Study of Afghan Saffron. AISA (Afghanistan Investment Support Agency), Kabul. Ketterings, Q.M., Albrecht, G., Czymmek, K., Bossard, S., 2005. Nitrogen Credits From Manure. Fact Sheet 4, Nutrient Management Spear Program. Sciences Cornell University, College of Agriculture and Life, Department of Crop and Soil Sciences, New York. Khorramdel, S., Gheshm, R., Amin Ghafori, A., Esmaielpour, B., 2014. Evaluation of soil texture and superabsorbent polymer impacts on agronomical characteristics and yield of saffron. J. Saffron Res. 1, 120
135 (in Persian). Kirmani, N.A., Sofi, J.A., Bhat, M.A., Ansar-Ul-Haq, S., 2014. Sustainable saffron production as influenced by integrated nitrogen management in Typic Hapludalfs of NW Himalayas. Commun. Soil Sci. Plant Anal. 45, 653
668. Koocheki, A., Seyyedi, S.M., 2015. Relationship between nitrogen and phosphorus use efficiency in saffron (Crocus sativus L.) as affected by mother corm size and fertilization. Ind. Crop. Prod. 71, 128
137. Kumar, R., Singh, V., Devi, K., Sharma, M., Singh, M.K., Ahuja, P.S., 2009. State of art of saffron (Crocus sativus L.) agronomy: a comprehensive review. Food Rev. Int. 25, 44
85. Lage, M., Cantrell, C.L., 2009. Quantification of saffron (Crocus sativus L.) metabolites crocins, picrocrocin and safranal for quality determination of the spice grown under different environmental Moroccan conditions. Sci. Hortic. 121, 366
373. Lo´pez Rodrı´guez, F.N., 1989. Estudio histolo´gico de Crocus sativus L. Tesina de Licenciatura, Universidad Pu´blica de Pamplona, Pamplona, Espan˜a (in Spanish). McGimpsey, J.A., Douglas, M.H., Wallace, A.R., 1997. Evaluation of saffron (Crocus sativus L.) production in New Zealand. N.Z. J. Crop Hortic. Sci. 25, 159
168. Meek, B., Graham, L., Donovan, T., 1982. Long-term effects of manure on soil nitrogen, phosphorus, potassium, sodium, organic matter, and water infiltration rate. Soil Sci. Soc. Am. J. 46, 1014
1019. Passioura, J.B., 2002. Soil conditions and plant growth. Plant Cell Environ. 25, 311
318. Ranjbar, A., Emami, H., Khorassani, R., Karimi Karouyeh, A.R., 2016. Soil quality assessments in some Iranian saffron fields. J. Agr. Sci. Tech. 18, 865
878. Rezvani-Moghaddam, P., Khorramdel, S., Mollafilabi, A., 2015. Evaluation of soil physical and chemical characteristics impacts on morphological criteria and yield of saffron (Crocus sativus L.). J. Saffron Res. 3, 188
203 (in Persian). Russell, E.J., 1912. Soil Conditions and Plant Growth. Longmans, London. Ryan, J., Ibrikci, H., Sommer, R., McNeill, A., 2009. Nitrogen in rainfed and irrigated cropping systems in the Mediterranean Region. Adv. Agron. 104, 53
104. Sampathu, S.R., Shivashankar, S., Lewis, Y.S., Wood, A.B., 1984. Saffron (Crocus sativus L.)—cultivation, processing, chemistry and standardization. CRC Crit. Rev. Food Sci. Nutr. 20, 123
157. Sepaskhah, A.R., Yarami, N., 2009. Interaction effects of irrigation regime and salinity on flower yield and growth of saffron. J. Hortic. Sci. Biotechnol. 84, 216
222. Skinner, M., Parker, B.L., Ghalehgolab Behbahani, A., 2017. Saffron Production: Planting Depth and Density for Saffron Corms. North American Center for Saffron Research and Development Burlington, Vermont, USA. Skrubis, B., 1990. The cultivation in Greece of Crocus sativus L. In: Tammaro, F., Marra, L. (Eds.), Proceedings of the International Conference on Saffron (Crocus sativus L.). L’Aquila, Italy, pp. 171
182, 27
29 October 1990. Srivastava, R., Ahmed, H., Dixit, R., Dharamveer, K., Saraf, S.A., 2010. Crocus sativus L.: a comprehensive review. Pharmacogn. Rev. 4, 200
208. White Book Saffron in Europe, 2006. Problems and strategies for improving the quality and strengthen competitiveness. Available from: ,http:// www.europeansaffron.eu/archivos/White%20book%20english.pdf..

Chapter 6

Saffron water requirements Alireza Koocheki1, Hamid-Reza Fallahi2 and Majid Jami-Al-Ahmadi2 1

Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran, 2Department of Agronomy and Plant

Breeding, Faculty of Agriculture, University of Birjand, Birjand, Iran

Chapter Outline 6.1 Introduction 6.2 Crop coefficients and potential evapotranspiration 6.3 Irrigation scheduling 6.3.1 Before corm lifting 6.3.2 After corm planting 6.3.3 Preflowering irrigation in autumn 6.3.4 During vegetative growth 6.3.5 Summer irrigation 6.4 Rainfed saffron production 6.5 Factors affecting water requirements 6.6 Irrigation methods

6.1

67 67 70 70 70 71 72 78 80 80 81

6.7 Water quality 6.7.1 Water quality parameters 6.7.2 Salt stress in saffron 6.8 Water-use efficiency and productivity 6.9 Physiological responses of saffron to water stress 6.10 Beneficial water-related approaches for saffron production 6.11 Saffron response to flooding 6.12 Indigenous irrigation knowledge 6.13 Conclusion References

82 82 83 83 84 85 87 87 88 88

Introduction

There is a big gap between potential and actual yield of saffron in many parts of its distribution areas in the world (Behdani and Fallahi, 2015; Jabbari et al., 2017). One reason for this gap is related to its inappropriate irrigation management. Reduced precipitation (depletion of water resources in particular), decrease in the amount of water harvesting from Qantas (underground conduit), and more reliance on groundwater and deterioration of water quality (in particular raised water salinity levels) are the main causes of reduced saffron yield associated with irrigation (Behdani and Fallahi, 2015; Koocheki et al., 2017). Nevertheless, saffron is still considered to be an appropriate crop for low-input systems of arid and semiarid areas, since plant production in these areas are generally water-limited (Azizi-Zohan et al., 2008). Saffron, as a summer dormant and winter active plant, is one of the most efficient crops in terms of water consumption (Fallahi et al., 2014). It is a small and bulbous plant with a growing season from midfall to midspring, which coincides with the rainy season. The lifecycle of saffron, which begins with flowering and subsequent aboveground vegetative growth, is dependent on fall rains or irrigation, along with decreasing temperatures. The growth period of saffron ends in spring after the production of replacement (newly formed) corms at about 220 days from the first irrigation. This timing is similar in all areas of saffron production around the world (Behdani et al., 2016; Koocheki et al., 2019). During this period, much of the water needed by this plant is supplied by rainfall (Fallahi et al., 2017a). Indeed, saffron has a low water requirement as only 3000
3600 m3 ha21 of water is used per year for saffron production in Iran (Fallahi and Mahmoodi, 2018a; Gresta et al., 2008). Despite the low water requirement of saffron, it responds positively to higher water availability in terms of corm growth and flower production (Fallahi et al., 2017a).

6.2

Crop coefficients and potential evapotranspiration

Determination of crop water requirements is required for irrigation scheduling, proper design of irrigation systems, and water resource planning (Azizi-Zohan et al., 2008). The water requirements of saffron, like other crops, are calculated based on the crop coefficient (Kc) and reference evapotranspiration (ETo). If ETo is known, saffron evapotranspiration Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00006-X © 2020 Elsevier Inc. All rights reserved.

67

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TABLE 6.1 Reference evapotranspiration (ETo), crop evapotranspiration (ETc), and crop coefficients (Kc) of saffron for different months in a 2-year-old farm. Month

ETo (mm day21)

Class A pan evaporation (mm day21)

ETc (mm day21)

Kc

Kp

October

2.6

4.7

1.2

0.45

0.3

November

1.8

3.3

0.9

0.50

0.3

December

1.3

1.8

0.8

0.65

0.4

January

1.0

2.8

0.7

0.75

0.4

February

1.0

2.3

0.8

0.75

0.4

March

1.4

2.9

1.0

0.75

0.4

April

2.6

2.8

2.0

0.75

0.5

May

3.4

6.2

2.0

0.60

0.3

Source: From Alizadeh, A., 2006. Irrigation. In: Kafi, M., Koocheki, A., Rashed-Mohassel, M.H., Nassiri, M. (Eds.), Saffron, Production and Processing. Science Publishers, New Hampshire, USA.

(ETc) can be determined by Eq. (6.1), but when only the pan evaporation data are available, ETc is calculated using Eq. (6.2) (Alizadeh, 2006). ETc 5 Kc 3 ETo

(6.1)

ETc 5 Kp 3 ðEp Þ

(6.2)

where ETc, Kp, and Ep are the consumptive saffron water use, pan coefficient, and pan evaporation, respectively. Before applying Eq. (6.2), the saffron pan coefficient should be obtained based on field experiments. In a 3-year study conducted by Alizadeh (2006), the saffron evapotranspiration rate compared to reference crop and class A pan evaporation during growth season (October
May) were determined and then monthly values of Kc and Kp calculated. The highest Kc was observed during January through to March and Kp reached to its maximum in March (Table 6.1). In the third growing season (3-year-old field), Kc increased by 16% compared with the second year of growth. Finally, the Kc curve of saffron was generated as illustrated in Fig. 6.1. Azizi-Zohan et al. (2008) in a study in the semiarid region of Fars province (29 N, 52 E, and 1810 m with semiarid climate) reported that the values of Kc varied through the growing season from 0.22
0.24 to 0.94
1.05, and 0.68
0.78 at the beginning, middle, and end of saffron growth cycle, respectively, for the first and second growth seasons. The values of potential evapotranspiration (ETcp) and ETo were 486 and 991 mm for the first year and 670 and 975 mm for the second year, respectively. Higher ETcp in the second growing season was a result of more newly formed corms produced by corm multiplication during previous growing season (Fig. 6.2). In another study conducted in the same province (Bajgah village, Shiraz, Fars province, Iran), the potential evapotranspiration and single and dual crop coefficients for saffron were calculated during the first and the second growing seasons. The potential evapotranspiration values were 523 mm for the first and 640 mm for the second growing seasons. The maximum evapotranspiration rates were 4.5 and 6.1 mm day21 in the first and second growing cycles, respectively. Kc values for the initial, middle, and late growth periods were 0.41, 0.93, and 0.29 in the first year, and 0.45, 1.05, and 0.31 in the second year, respectively (Yarami et al., 2011). In this province, the potential evapotranspiration and crop coefficient of saffron also have been studied for the third and fourth growing seasons. The total values of potential evapotranspiration were 726 and 783 mm for the third and fourth growing seasons, respectively. The maximum potential evapotranspiration rates were 6.28 and 6.4 mm day21 for the third and fourth years, respectively. The single crop coefficient for the initial, middle, and the final growth stages were 0.46, 1.2, and 0.35 in the third year, and 0.49, 1.25, and 0.35 in the fourth year, respectively. Basal crop coefficients for the third stages of the growth in the third and the fourth years were 0.15, 0.9, and 0.17 and 0.15, 0.95, and 0.18, respectively (Keykhamoghadam et al., 2013). The Kc curve for a 3-year-old saffron field in the semiarid climate of Fars province is shown in Fig. 6.3 (Sepaskhah and Kamgar-Haghighi, 2009). In another research conducted in Isfahan province (30
34 N, 49
55 E), the Kc of saffron for initial (30 days, starting from 1 October), development (55 days), middle (105 days), and last (30 days, ending on 10 May) growth phases

Saffron water requirements Chapter | 6

69

FIGURE 6.1 Crop coefficient curve of saffron during different growth stages for two growing seasons in 2- and 3-year-old farms. From Alizadeh, A., 2006. Irrigation. In: Kafi, M., Koocheki, A., Rashed-Mohassel, M.H., Nassiri, M. (Eds.), Saffron, Production and Processing. Science Publishers, New Hampshire, USA.

FIGURE 6.2 Crop coefficient curves of saffron during different growth stages for two growing seasons. From Azizi-Zohan, A.A., Kamgar-Haghighi, A.A., Sepaskhah, A.R., 2008. Crop and pan coefficients for saffron in a semi-arid region of Iran. J. Arid Environ. 72, 270
278.

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SECTION | II Safron production

FIGURE 6.3 Crop coefficient for a 3-year-old saffron field at different growth stages. From Sepaskhah, A.R., KamgarHaghighi, A.A., 2009. Saffron irrigation regime. Inter. J. Plant Prod. 3(1), 1
16.

were considered as 0.41, 0.65, 0.85, and 0.55, respectively. The crop water requirement for the initial growth stage varies between 33 and 68 mm in different counties. The range of water requirement was 38
85 mm for the developmental stage, 140
265 mm for the middle stage, and 68
116 mm during the last growth phase. Accordingly, the saffron water requirement during the whole growth period was estimated to be 279
528 mm, while rainfall ranged from 82 to 262 mm across the study area (Rajabi et al., 2015). In a study in the Pampore area of Kashmir Valley, India (34 N, 74 E, and elevation of 1574 m), the water requirements of saffron were found to be 288 mm during a whole season growth. This amount for initial (sprouting to flowering), middle (vegetative growth period), and late season were 80, 134, and 75 mm, respectively (Ahmad et al., 2017).

6.3

Irrigation scheduling

6.3.1 Before corm lifting Saffron corms can be lifted from the soil of old flowering fields or corm production fields during real (after leaves senescence in midspring to end of June) or pseudo-dormancy (from early July up to early-autumn) stages, manually or by mechanical tools. Although the best time to do this is during the real dormancy period, in traditional production of saffron it is more common in the pseudo-dormancy phase (Behdani et al., 2018). Some farmers irrigate the old fields once, then move on to corm lifting to facilitate corm harvesting when corms must be planted in new fields immediately after lifting. However, this is not recommended due to the need to keep corms in their summer dormant state and to avoid infection of possible soilborne diseases (Behdani and Fallahi, 2015).

6.3.2 After corm planting The effects of irrigating immediately after corm planting have not been fully investigated in saffron. In a 2-year field experiment, the combined effects of planting date (22 of May, July, and September) and irrigation management (irrigation and no irrigation after each planting date) were studied on saffron flowering. All studied flowering criteria were superior in nonirrigated treatment after planting at all three planting dates (Table 6.2). On average in all planting dates, the flower yields in the first and second growing seasons were 9 and 43 kg ha21 with irrigated treatment and 37 and 78 kg ha21 with nonirrigated treatment, respectively (Koocheki et al., 2016a). Accordingly, it has been recommended that saffron be planted during the real dormancy stage (i.e., in the late spring), but its first irrigation should be postponed until midfall, when providing moisture in the soil along with lowering the air temperature leads to the emergence of flowers (Behdani et al., 2018). Irrigation management after corm planting can also affect the corm growth characteristics of saffron. Koocheki et al. (2016b) reported that irrigation immediately after corm planting increased the amount of contractile roots and number of replacement (daughter) corms per plant (12%), but decreased the amount of the mean number of flowering buds (19%), mean number of total buds (11%), and mean weight (19%), and diameter (8%) of replacement corms. However, the response to irrigation was strongly dependent on planting date, where irrigation after corm planting in spring and summer negatively affected corm growth, whereas autumn irrigation (23 September) favored corm growth. In another study, it was found that when corm planting in late spring or midsummer it is better to avoid immediate irrigation after planting to obtain better leaf growth and aboveground biomass yield (Koocheki et al., 2011).

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71

TABLE 6.2 Effect of irrigation immediately after corm planting on saffron flowering at three planting dates. Year

Planting dates

Irrigation

2009

22 May

I NI

22 July

2010

22 May

22 July

22 September

4.3a* a

15.2

Flower yield (kg ha21)

Stigma dry yield (g ha21)

Flowers per plant

Flowering rate (day21)

10.5a

135a

0.10a

1.4c

a

a

590

a

0.38

7.1a

45.4

4.0a

8.7a

120a

0.09a

1.4c

14.3a

36.7a

480a

0.35a

5.9a

3.5a

7.8a

120a

0.08a

NI

11.7a

28.0a

419a

0.29a

3.4b

I

12.6c

39bc

615bc

0.31bc

3.6bc

NI

48.0a

127a

1675a

1.2a

d

c

c

I NI

22 September

No. flower per m2

I

1c

15.8a

7.1

25

445

0.18

2.4c

NI

26.8b

75b

1039b

0.67b

8.9b

I

10.6c

34c

554c

0.26bc

3.8bc

7.8d

27c

463c

0.19c

2.1c

I

NI

c

I, Irrigation; NI, no irrigation. In each column, means with at least one similar letter are not significantly different at 5% level of probability. Source: From Koocheki, A., Rezvani-Moghaddam, P., Fallahi, H.R., 2016a. Effects of planting dates, irrigation management and cover crops on growth and yield of saffron (Crocus sativus L.). J. Agroecol. 8(3), 435
451 (in Persian).

6.3.3 Preflowering irrigation in autumn The first irrigation time in fall is a critical determinant of the yield and quality of saffron. This plant can be synanthous (leaves appear before flowers) or hysteranthous (leaves appear after flowering), depending on the first irrigation timing (Yasmin and Nehvi, 2018). On-time irrigation allows flowers to appear before leaves emerge, which makes flower picking easier. Early irrigation accelerates the leaf growth, while with delayed irrigation the flowers may be exposed to chilling damage (Behdani and Fallahi, 2015; Menia et al., 2018). The start of flower emergence and the first irrigation are quite different in different regions and are strongly dependent on weather conditions, especially temperature. Flowering occurs when air temperatures reach around 12 C (Alizadeh et al., 2009). Given that flowering starts 2
3 weeks after irrigation, the first irrigation should be applied at an appropriate time so that the thermal requirement of flowering is met during this period. In this case, after passing this time course, the flowers are ready to be picked. In areas with higher elevation and lower daily air mean temperature, the dates of first irrigation and flowering usually occur in the early fall, while decreasing elevation and increasing daily air temperature leads to delay in flowering date and first irrigation time up to the middle or even late autumn (Alizadeh et al., 2009). In a study in Isfahan province, Iran, the optimum flowering date of saffron, based on weather conditions, was determined to be from the first half of October to the late of November. By considering the flowering date and daily temperature requirement of saffron, the preflowering irrigation date ranges from the second half of September in the western parts to early November in the eastern regions (Fallah Ghalhary and Ahmadi, 2015). Sepaskhah and Kamgar-Haghighi (2009) also reported that the time of preflowering irrigation has a significant effect on the flowering and yield of saffron. They concluded that under Mashhad (northeast of Iran) climatic conditions both early and late first preflowering irrigations limit saffron flowering considerably (Table 6.3). In Kashmir, India saffron needs weekly preflowering irrigations during September and October. In this region, 100
150 mm rainfall is helpful through the preflowering stage, but irrigation can improve the yield by 40% if there is no rainfall (Husaini, 2014; Taufique et al., 2017). Shah and Tripathi (2008) also emphasized sprinkle irrigation of saffron fields with intervals of 15 days during August to September, when there is no summer rain in Kashmir. In a similar study, Ahmad Aga (2008) reported that the highest yield of saffron in Kashmir was obtained when the field was irrigated five times at 10-day intervals from 20 August to 30 September, with 760 m3 ha21 of water within the specified time period.

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SECTION | II Safron production

TABLE 6.3 Saffron yield as influenced by time of preflowering irrigation. Flower dry weight (g m22)

Time of first irrigation

Saffron yield (g m22)

Early October

8.25

0.156

Mid-October

11.02

0.226

Late October

8.47

0.170

Mid-November

6.74

0.114

Source: From Sepaskhah, A.R., Kamgar-Haghighi, A.A., 2009. Saffron irrigation regime. Int. J. Plant Prod. 3(1), 1
16.

TABLE 6.4 Response of saffron yield to the date of first autumnal irrigation. First irrigation date

Number of flower per m2

Flower yield (kg ha21)

Corm yield (g m22)

Mean replacement corm weight (g)

Number of largesized corms per m2

Leaf length (cm)

7 October

19.4b

62.2b

531.3a

6.34a

6.13b

21.2a

22 October

25.8a

92.6a

543.0a

6.42a

5.67b

16.2b

6 November

20.8b

73.0b

563.5a

6.58a

8.00a

14.3c

In each column, means with at least one similar letter are not significantly different at 5% level of probability.

Source: Data from Osmani-Roudi, H.R., Masoumi, A., Hamidi, H., Razavi, S.A.R., 2015. Effects of first irrigation date and organic fertilizer treatments on saffron (Crocus sativus L.) yield under Khaf climatic conditions. Saffron Agron. Technol. 3(1), 25
33 (in Persian).

Under the climatic conditions of Khorasan, the largest saffron producer province of Iran, mid-October is the best time for the first irrigation of saffron fields (Alizadeh, 2006). Mohammad Abadi et al. (2011) tested three dates (22 August, 22 September, and 22 October) as start dates of saffron irrigation in Mashhad and observed no significant difference between treatments in terms of flower and corm yields, although crops irrigated on 22 September performed better. However, in another study in the same area the first irrigation in early November was more effective in improving corm growth and saffron flowering (Rezvani-Moghaddam et al., 2011). This difference in time of the first irrigation in various reports can be partly attributed to fluctuations in temperature from year to year. In a study in Khaf county in Khorasan Razavai province (34 N, 60 E, 950 masl) the response of saffron flowering and corm production on three dates of first autumnal irrigation (7 October, 22 October, and 6 November) were evaluated. The highest number of flowers, flower yield, and number of small-sized corms (4
6 g) (40.9 corm m22) were obtained from the 22 October irrigation date, while corm yield, mean weight of corm, corm number (85.1 corm m22), and number of middle-sized (6
8 g) (22.8 corm m22) and large-sized corm ( . 8 g) (8 corm m22) were obtained when the first irrigation was applied on 6 November. The highest number of very small-sized corms (,4 g) and leaf length were observed when the first irrigation was applied on 7 October (Table 6.4) (Osmani-Roudi et al., 2015). In late autumnal irrigation, crust breaking should be avoided or done with great care, because prolonged buds are close to the surface ground, thereby crust breaking may damage them. After primary irrigation, farmers soften the soil surface by different mechanical implements, which will facilitate the upraise of saffron flowers from the subsurface. It has been reported that with late irrigation, the highest yield is obtained with manual or no crust breaking due to considerable growth of sprouts up to the near surface of soil. Economical evaluation also revealed that using a rotavator is the best method for early and on-time irrigation, and the suitable method for late irrigation is no crust breaking (Saeidirad et al., 2007). Crust breaking is useful for easier flower emergence especially in soils with unsuitable structure, low organic matter or heavy texture. (Behdani and Fallahi, 2015).

6.3.4 During vegetative growth There is a close relationship between water availability and saffron yield (Fig. 6.4). The water requirements of saffron is different during and between growth seasons. Traditionally, saffron is irrigated four to five times during vegetative growth stage, although more water consumption during October
May increases the yield (Behdani et al., 2015). In

Saffron water requirements Chapter | 6

73

FIGURE 6.4 Effect of irrigation intervals on saffron stigma yield in Khorasan province of Iran. From Koocheki, A., Behdani, M.A., NassiriMahallati, M., 2006. Agronomic attributes of saffron yield at agroecosystems scale in Iran. J. Appl. Hort. 8(2), 121
124.

TABLE 6.5 Corm and stigma production of saffron in different irrigation intervals. Irrigation intervals

Stigma yield (kg ha21)

Aboveground biomass yield (kg ha21)

No. of large corm ( . 8 g) per m2

Corm yield (tons ha21)

First growing season

Second growing season

12 days

1.64a

3.95a

644a

55.0a

13.2a

24 days

1.65a

2.93b

602ab

34.7b

9.5b

36 days

1.11b

1.85c

473bc

40.3ab

8.9b

Rainfed

0.44c

0.58d

386c

8.9c

3.1c

Source: Data from Azizi-Zohan, A.A., Kamgar-Haghighi, A.A., Sepaskhah, A.R., 2006. Effect of irrigation method and frequency on corm and saffron production (Crocus sativus L.). J. Water Soil Sci. 10(1), 45
54 (in Persian).

general, when seasonal rainfall is around 400 mm, more deficit irrigation (50% ETp) is allowed, while, with a seasonal rainfall of about 200 mm, lower deficit irrigation (75% ETp) is recommended (Sepaskhah and Kamgar-Haghighi, 2009). Saffron has a shallow root system and therefore cannot absorb water from the deep layers of soil. Accordingly, it is better to increase the irrigation frequencies by reducing the volume of water used in each irrigation, instead of less frequent heavy irrigation (Behdani and Fallahi, 2015). In a study in Shiraz (25 N, 52 E, 1810 masl) the highest stigma yield in the first growth season was obtained with irrigation with a 24-day interval (380 mm water consumption during plant growth), while in the second growth season, irrigation intervals of 12 days (650 mm water during plant growth) were more suitable (Table 6.5). Moreover, irrigation treatments showed significant differences when compared with the rainfed farming system, which received 430 mm rainfall 1 180 mm supplementary irrigation water during the first growth seasons and only 200 mm rainfall during the second growth season (Azizi-Zohan et al., 2006). ShahriariAhmadi (2016) in a study on saffron in Mashhad reported that a 3-week irrigation interval produced more flower and stigma yield than 1- and 2-week intervals (Table 6.6). Fallahi and Mahmoodi (2018b) obtained similar results in a field study in a dry region of Southern Khorasan province (110 mm annual rainfall), where mean weights of replacement corms in 14- and 28-day irrigation intervals were 5.4 and 6.5 g, respectively. They stated that annual application of 3600 m3 ha21 water plus cow manure is an appropriate strategy for saffron production in semiarid regions. A 9-year assessment of four irrigation regimes (50%, 75%, and 100% ETo and rainfed) for saffron revealed that supplying more water caused more corm numbers and corm yield per hectare during all studied years (Khazaei et al., 2013). Water availability can also affect the amount of nutrient uptake. When the effects of 50%, 75%, and 100% of saffron water requirements (523 and 640 mm during the first and the second growth cycles, respectively) were evaluated on nitrogen acquisition by crop, it was found that supplying 75% of the water requirement was the best treatment with respect to nitrogen uptake indices (Table 6.7) (Koocheki et al., 2014a). The same treatments were also used to evaluate the growth and phosphorus uptake of daughter corms. More water availability improved the corm number, yield, and phosphorus content of corm. In the first growing season, flowering traits were not affected when 50% of the

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SECTION | II Safron production

TABLE 6.6 Flower and stigma yield of saffron under several irrigation intervals. Irrigation intervals

Flower yield (g m22)

No. flower per m2 Second growing season

Dry stigma yield (g m22)

Third growing season

Second growing season

Third growing season

Second growing season

Third growing season

1 week

97.1c

168.2b

45.9a

64.6b

0.50b

1.0b

2 weeks

110.5b

189.0ab

46.5a

71.9ab

0.57a

1.1ab

3 weeks

124.2a

203.7a

42.2b

74.6a

0.62a

1.2a

a

c

a

1.1a

4 weeks

109.9

bc

207.7

ab

36.1

66.9

0.59

In each column, means with at least one similar letter are not significantly different at 5% probability level.

Source: From Shahriari-Ahmadi, R., 2016. Effect of nutrient management, wheat straw and irrigation interval on flower and corm yields of saffron (Crocus sativus L.) under Mashhad ecological environment (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

TABLE 6.7 Relationship between water availability and nitrogen uptake in saffron. Water availability (% of water requirement)

Growing season

Nitrogen content (g m22) Corms

50

75

100

Nitrogen uptake efficiency (%)

Nitrogen use efficiency (g g21)

Nitrogen harvest index (%)

leaves

e

5.96e

22.14e

12.24e

53.97c

Second

10.06d

7.40d

28.81d

16.47d

56.47d

First

16.82c

8.08c

40.59c

27.14c

64.84b

a

b

a

57.65

a

40.59

69.74a

First

7.54

Second

24.81

10.22

First

16.52c

8.22c

40.46c

26.79c

65.21b

Second

21.76b

10.70a

53.65b

35.78b

66.28b

In each column, means followed by at least one letter in common are not significantly different at the 5% probability level using Duncan’s multiple range test.

Source: From Koocheki, A., Seyyedi, S.M., Jamshid-Eyni, M., 2014a. Uptake efficiency of nitrogen in saffron (Crocus sativus L.) as affected by irrigation levels and high corm density. Seed Plant Prod. J. 30(4), 441
456 (in Persian).

crop water requirement was met. In the second year, however, flower number, flower yield, and dry stigma yield decreased by supplying 50% (by 19%, 28%, and 22%, respectively) as compared to 100% of the water requirement (Table 6.8). Overall, with regard to the high value of water in arid and semiarid regions, the irrigation scheduling based on supplying 75% of the crop water requirement was considerable (Koocheki et al., 2014b). In another study the combined effects of fertilization and two levels of irrigation (7- and 14-day irrigation intervals during plant growth equal to 3600 and 7200 m3 ha21 water, respectively) were studied on saffron yield and chlorophyll fluorescence parameters. In low-watered plants, applying manure produced a higher Fv/Fm ratio than those with no fertilizer treatment, but no significant effect was observed in well-watered plants by manure application. Lower water availability did not impose any limitation on the photosynthetic process of saffron. Appropriate saffron performance under lower water availability treatment is due to its special leaf anatomy and stomatal structure, which leads to low water requirements, especially during winter (Fallahi et al., 2017c). However, from the early spring up to the leaf senescence of saffron, the water availability in intervals of 12 days has an increasing effect on replacement corm size and flowering in the coming season (Behdani and Fallahi, 2015). Based on the above, high water availability may not necessarily be beneficial to saffron. Mosaferi-Ziaoddini (2001) also tested four levels of applied water (10, 20, 40, and 80 mm corresponding to 100, 200, 400, and 800 m3 ha21, respectively) with 15-day intervals during a 6-month growth season of saffron in the Mashhad area. Application of 20 mm water at each irrigation time (equivalent to about 240 mm of seasonal applied water) resulted in the highest saffron yield (Table 6.9). Therefore it seems that although water stress is a limiting factor for saffron growth, overirrigation also is not useful, particularly in areas with low water resources.

Saffron water requirements Chapter | 6

75

TABLE 6.8 Effect of water availability on flower and stigma yield and phosphorous uptake by replacement corms in saffron. Water availability (% of water requirement)

Growing season

Flower yield (g m22)

Stigma yield (mg m22)

Phosphorus concentration in replacement corms (g kg21)

Phosphorus content of replacement corms (g m22)

50

First

25.5b

196.9b

1.63a

1.13b

b

b







Second 75

100

27.2

207.1

b

b

ab

First

27.5

205.8

1.50

2.34a

Second

36.6a

272.0a







b

b

b

First

28.9

217.5

1.46

2.32a

Second

37.8a

264.1a







In each column, means with at least one similar letter are not significantly different at 5% probability level. Source: From Koocheki, A., Seyyedi, S.M., Jamshid-Eyni, M., 2014b. Effect of irrigation levels and high corm density on growth and phosphorus uptake of daughter corms of saffron (Crocus sativus L.). Iran. J. Crop Sci. 16(3), 222
235 (in Persian).

TABLE 6.9 Saffron yield as affected by different amounts of applied water at each irrigation date. Applied irrigation water (mm)

Flower dry weight (g m22)

Saffron yield (g m22)

10

25.0b

0.150b

20

a

37.0

0.190a

40

34.0ab

0.153b

80

27.1b

0.117b

In each column, means with at least one similar letter are not significantly different at 5% probability level. Source: From Sepaskhah, A.R., Kamgar-Haghighi, A.A., 2009. Saffron irrigation regime. Int. J. Plant Prod. 3(1), 1
16.

In a study in Birjand (32 N, 59 E, 1490 masl) the saffron water requirement, determined by FAO-Penman-Monteith equation, was estimated to be 2350 m3 ha21 during the growth season. The measured irrigation water used by saffron farmers was about 1184 m3 ha21 (Ahmadee et al., 2017), which indicates they are already applying deficit irrigation techniques based on their traditional knowledge. The highest water requirement of saffron occurs in the mid- and late season (Fallah Ghalhary and Ahmadi, 2015). Accordingly, in a study on the effects of irrigation intervals (2, 3, and 4 weeks equal to 1800, 2400, and 3000 m3 ha21) from 6 March up to the leaf senescence stage, it was concluded that more water availability in the late growing season is crucial for better corm growth and flower yield of saffron in the coming season (Table 6.10) (Aghhavani-Shajari, 2017). In response to providing 100%, 75%, and 50% of the water requirements a study of the vegetative growth parameters of saffron in Ashkzar (Yazd province, Iran) showed that there was not a significant difference between 100% and 75% treatments. When 50% of the water requirement was supplied, some reduction in the growth parameters were observed (Gholami et al., 2017), which shows the low water demand of saffron plants (Table 6.11). Nehvi et al. (2007) reported that instead of rainfed saffron production under Kashmir conditions, India irrigation during September
December is preferred to achieve higher productivity. They found that providing water during September
October (preflowering) and November
December (postflowering) through sprinklers at 700 m3 ha21 increased saffron yield by up to 40%. In this region, saffron fields are rainfed (1000
1200 mm per year) with low productivity in dry years (Kumar et al., 2009). For example, a drought period during 1999
2003 caused a severe reduction in stigma yield from 3.12 to 1.57 kg ha21. Therefore, 10 times sprinkler irrigation with weekly intervals, each time 70 m3 ha21, during pre- and postflowering stages helps corm sprouting, flower emergence, and finally increases yield (Husaini et al., 2010).

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SECTION | II Safron production

TABLE 6.10 Effect of irrigation management at the late growth season on saffron performance in Mashhad. Water application (m3 ha21)

Flower yield (kg ha21) 1

Dry stigma yield (kg ha21)

2014 a

ab

2015

2014

a

a

Corm yield (g m22) 2015

Mean replacement corm weight (g)

2014 a

a

2015

2014

a

3000

108.8

308.2

1.27

3.80

2457

3855

2.84

3.31a

2400

117.2a

278.5b

0.66a

3.29a

1764b

2995b

2.96a

3.08ab

1800

55.1b

392.4a

0.91a

3.44a

1606b

2855b

1.93b

2.56b

In each column, means with at least one similar letter are not significantly different at 5% probability level. Source: From Aghhavani-Shajari, M., 2017. Study the Possibility of Increasing Saffron (Crocus sativus L.) Corm Size and Flower Yield Through the Farming Managements (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

TABLE 6.11 Mean of saffron measured parameters under different irrigation treatments in the first year. Treatments (% ET)

Weight of the biggest corm (g)

Total replacement corm weight per plant (g)

Relative water content of leaf

Corm number per plant

Leaf length (cm)

50

5.35b

6.92a

0.68b

2.04a

36.35b

75

6.16a

7.42a

0.72ab

1.76a

39.18a

100

6.47a

7.31a

0.75a

1.67a

39.38a

Within each column, same letters indicate no significant difference between treatments (P , .05). In each column, means with at least one similar letter are not significantly different at 5% probability level. Source: Data from Gholami, M., Kafi, M., Khazaei, H.R., 2017. Study of the relations of sink and source in saffron by means of correlation coefficients under different irrigation and fertilization levels. Saffron Agron. Technol. 5(3), 195
210 (in Persian).

In Morocco, Taliouine (30 N, 8 W, 1200
1630 masl) is the main saffron production region, with 100
300 mm annual rainfall. In a study in the area, irrigation was started in early September and applied every 2 weeks until the flowering stage. After flowering, two to three irrigations were applied up to the end of the growing cycle depending on rainfall distribution (Lage and Cantrell, 2009). In this region, 350
500 m3 ha21 water is applied in weekly intervals from September
November and every other week from December
March, while no irrigation is given during corm dormancy of saffron (April
August) (Ait-Oubahou and El-Otamani, 1999). In Morocco, fields have to be irrigated regularly in the vegetative period, with weekly intervals around the flowering periods, because irrigation is crucial when there is no rain (Filipski et al., 2017). In Taliouine, Morocco, the amount of water used in saffron fields depends heavily on rainfall (B317 mm annually). In this region the critical periods for water requirements are flowering (in October) and the replacement corm growth phases (March
April), which require one or two irrigations per month. It has been reported that under Morocco’s climatic conditions, when there is no rainfall, irrigation is applied 2 weeks before flowering and twice at the beginning of flowering (mid-October) (Aziz and Sadok, 2015). In Turkey, a dry summer after a rainy period in spring is appropriate for saffron production. In a study in Karabuk, Turkey, it was reported that 300 mm annual precipitation is required for saffron production. Therefore, annual rainfall of 440 mm in Karabuk is suitable in terms of ecological demands of the plant (Coskun et al., 2017). In Spain, irrigated saffron production is performed in dry temperate regions with an annual rainfall of 400 mm (Kumar et al., 2009). In Greece, saffron is grown in Kozani county with an annual rainfall of 500 mm, and the plant has two critical stages when moisture availability in soil, by irrigation or rain, is indispensable: one during September for best flowering and another during March and April, when replacement corms grow rapidly (Goliaris, 1999). In Navelli, Italy, irrigation is not necessary for saffron production (Tammaro, 1999). Under Afghanistan conditions, the rainfall requirement for appropriate saffron growth is about 300 mm annually. In addition, the maximum water requirement of the plant is March
April with 150
200 m3 ha21 needed during each irrigation round (Katawazy, 2013). In a preliminary study in Uzbekistan (Fergana and Tashkent cities), it was reported that one irrigation is necessary for the beginning of vegetation (preflowering). After that, another irrigation is useful during

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budding or flowering, but in dry soils. However, postflowering irrigation is not required until the end of the growing season (Valijonovich, 2018). In general, at least four or five irrigations are necessary for proper saffron production in arid and semiarid regions of Iran. The first irrigation is done in early October to induce corm sprouting and flower emergence (locally known as Besar-Ab or Gol-Ab). The second one in November after flowers are picked up and leaf appearance (locally known as Zaeech-Ab), and the third one is made after winter weeding during late December to early January (locally known as Kulesh-Ab). It must be noted that winter irrigation during cold periods or cold areas must be avoided due to freezing risks. After the third irrigation, another irrigation is applied in early March and again in April to allow replacement corms to complete their growth (locally known as Zard-Ab). Moreover, in some cases irrigation is recommended in the middle of summer to induce flowers. In some areas, the third and fourth irrigation rounds are ignored due to sufficient winter precipitation (Koocheki et al., 2016d; Behdani et al., 2018). For appropriate weed control, it is better to postpone irrigation after weeding during plant growth for about 2 weeks based on field observations (Behdani and Fallahi, 2015). In one study, the effect of four irrigation treatments on quality and quantity of saffron was studied. The maximum flowering, corm production, and stigma yield were observed when at least four irrigations were applied during the saffron growth cycle. Under water-deficit conditions, crocin and picrocrocin content and phosphorus concentration were increased, possibly to help plants to cope better with water stress (Table 6.12) (Koocheki et al., 2016d). The combined effect of water and nutrient availability in dry regions is also an important factor determining corm growth and flowering of saffron. In an experiment, the effects of irrigation regimes [2- and 4-week intervals equal to application of 3600 m3 ha21 (conventional irrigation treatment) and 7200 m3 ha21 (overirrigation treatment)], combined with 30 tons ha21 cow manureand chemical fertilizers (220, 150, and 100 kg ha21 urea, superphosphate and potassium sulfate, respectively) were studied. The total aboveground biomass during the last two growth months was higher for cow manure treatment in both levels of water availability. The highest crop growth rate (12 g m22 day21) was obtained 145 days after first autumnal irrigation with cow manure treatment. The highest values of flower number (98 flowers m22), flower yield (24.3 g m22), style yield (0.56 g m22), and dry petal yield (3.7 g m22) were obtained in TABLE 6.12 Effects of irrigation rounds on corm, flower, and stigma yields, phosphorous uptake, and quality of stigma in saffron. Irrigation rounds

Flower number (m2)

Dried stigma yield (mg m22)

Number of daughter corms (m2)

Weight of daughter corms (g m2)

No irrigation

24.2e

55.4e

274b

789e

August

65.3d

200.2d

279b

922d

August 1 October

88.6c

292.3c

282ab

1097c

b

b

a

August 1 October 1 November

97.1

329.5

288

1193b

August 1 October 1 November 1 April

105.0a

353.9a

289a

1258a

Irrigation rounds

Phosphorus content in total plant (g m22)

UV
visible Crocin

(E 1% 440nm Þ

Picrocrocin (E 1% 275nm Þ

Safranal (E 1% 330nm Þ

No irrigation

2.38e

268.7a

94.7a

36.6a

August

2.83d

254.0b

85.2b

37.2a

August 1 October

3.24c

233.4c

72.3c

37.0a

b

d

c

August 1 October 1 November

3.64

207.2

72.7

37.5a

August 1 October 1 November 1 April

3.85a

207.5d

72.0c

37.1a

In each column and for each index, means with at least one similar letter are not significantly different at 5% level of probability. Source: From Koocheki, K., Ebrahimian, E., Seyyedi, S.M., 2016d. How irrigation rounds and mother corm size control saffron yield, quality, daughter corms behavior and phosphorus uptake. Sci. Hortic. 213, 132
143.

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FIGURE 6.5 Effect of fertilizer type and irrigation intervals on saffron flower yield. Similar letters are not significantly different at 5% level of probability, based on LSD test. From Fallahi, H. R., Mahmoodi, S., 2018a. Influence of organic and chemical fertilization on growth and flowering of saffron under two irrigation regimes. Saffron Agron. Technol. 6(2), 147
166 (in Persian).

FIGURE 6.6 Effect of fertilizer type and irrigation intervals on saffron style yield. Similar letters are not significantly different at 5% level of probability, base on LSD test. From Fallahi, H.R., Mahmoodi, S., 2018a. Influence of organic and chemical fertilization on growth and flowering of saffron under two irrigation regimes. Saffron Agron. Technol. 6(2), 147
166 (in Persian).

plants treated with cow manure that received water in 4-week intervals. The highest flower and stigma yield were obtained with irrigation intervals of 4 weeks and cow manure fertilizing and control treatments, and in 2-week irrigation intervals when chemical fertilizer was applied (Figs. 6.5 and 6.6). It appears that saffron water requirements increase under chemical fertilizer application. In addition, applying a maximum of 3600 m3 ha21 of water is sufficient during the first growth cycle of saffron, when corm density and transpiration area are low (Fallahi and Mahmoodi, 2018a). During each growing season replacement corms develop on the buds of the mother corm and thus the corm density and subsequently leaf area as transpiration surface will increases in coming seasons (Fallahi and Mahmoodi, 2018b). Crop models can also be used to determine the optimum irrigation leading to the best water productivity in saffron production. In a study, the AquaCrop model was investigated for simulating growth and yield of saffron under different irrigation regimes. Results revealed that the model could simulate the water content of different soil layers (NRMSE, normalized root mean square error 5 14%), potential evapotranspiration (NRMSE 5 27%), saffron yield (NRMSE 5 11%), and biomass production (NRMSE 5 25% for pooled over the 6 years). Accordingly, it suggested that the AquaCrop can be used for saffron yield simulation in all growing seasons (as a perennial crop), but it is more precise for biomass simulation only in earlier growing seasons with lower accuracy in the last years of growth (Mirsafi et al., 2016).

6.3.5 Summer irrigation In general, it is believed that summer irrigation promotes saffron flowering. However, sometimes it is not recommended because of the high risk of pests and fungal disease (Alizadeh, 2006). For example, it was observed that summer irrigation on 10 July and August significantly increased mite (Rhizoglyphus robini) population and reduced the yield. In the same study, applying two irrigations maximized the mite population in all planting depths. In general, the lowest levels of mite population as well as black spots on corms (symptoms of mite damage) were observed with no summer irrigation (Rahimi et al., 2008). Hence, summer irrigation is not recommended during hot periods and heavy soils (due to higher ability in water holding) to avoid corm infection (Behdani and Fallahi, 2015). In a study, continuing irrigation in the spring (after May) and summer (until August) significantly reduced the yield of saffron (Seghatoleslami and Sabzekar, 2017).

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TABLE 6.13 Effect of summer irrigation and tillage on stigma and corm yield of saffron. Treatments

No. flower per m2 Year 1

Year 2

Dry stigma yield (g m22)

Replacement corm yield (g m2)

Year 1

Year 1

Year 2

Summer irrigation c

c

July

16.5

84.9

0.098c

0.439b

898a

August

19.7b

127.4a

0.126b

0.968a

816a

July 1 August

25.0a

84.3c

0.157a

0.493b

855a

No summer irrigation

10.5d

98.8b

0.056d

0.555b

706b

Application

18.8a

99.8a

0.115a

0.620a

814a

a

a

b

a

824a

Tillage after summer irrigation

No application

17.0

98.0

0.103

0.600

Source: From Feizi, H., Mollafilabi, A., Sahabi, H., Ahmadian, A., 2015. Effect of summer irrigation and conservation tillage on flower yield and qualitative characteristics of saffron (Crocus sativus L.). Saffron Agron. Technol. 2(4), 255
263 (in Persian).

A summer irrigation date is highly dependent on climatic conditions and thus its appropriate date is variable between locations and years (Koocheki et al., 2016b). In an investigation it was observed that no irrigation during summer has a positive effect on the mean weight of replacement corms and flowering capacity of saffron (Koocheki et al., 2016a,b). However, leaving plant residue on the soil surface can positively affect the efficiency of summer irrigation (Koocheki et al., 2016b). In this regard, Amin-Amlashi et al. (2014) concluded that summer irrigation, especially when combined with planting squash as a cover crop, increased flowering and the quality of saffron. Some other researchers believe that soil tillage shortly after irrigation may partially improve the summer irrigation effectiveness (Feizi et al., 2015). They examined the combined effect of summer irrigation (6 July, 6 August, July 1 August, and no irrigation) and tillage a few days after irrigation on saffron corm and flower production during two growing seasons. In the first year, the highest number of flowers and flower and stigma yields of saffron were obtained with irrigating in July 1 August, while in the second year, irrigation in August had the highest significant positive effects. Also, the flower and stigma yields of saffron were significantly increased by tillage during the first growing season (Table 6.13). The positive effects of tillage and summer irrigation were related to reduced temperature and providing adequate humidity during the flower initiation stage in midsummer. The results of Koocheki et al. (2013) revealed that continuous summer irrigation has a positive influence on saffron growth if marjoram (Origanum majorana) is planted in the field during summer. The highest saffron yield (0.27 g m22) was obtained with irrigation every 14 days, while irrigation every 7 days caused the lowest yield (0.09 g m22). The maximum yield of saffron (0.20 g m22) was obtained by planting a combination of one row of saffron and one row of marjoram while the minimum yield was obtained with pure stand of saffron (0.15 g m22). It was concluded that intercropped saffron with marjoram increased the flower and stigma yields due to decreased soil temperature. Thus the planting method could be regarded as an alternative in saffron-based agroecosystems to deal with the possible impacts of soil warming, leading to better adaptation to climate change consequences (Koocheki et al., 2013). Summer irrigation may be effective in promoting corm growth and flower yield of saffron. However, it may be linked with irrigation date and planting depth. For example, in a long-term study in Gonabad (34 N, 58 E, 1105 msl) in Iran, a higher flower yield was obtained only with 10 cm planting depth (compared with 20 and 30 cm) with summer irrigation in July (compared with one irrigation in June and two irrigations, one in June and another in July). Nevertheless, summer irrigation may not be practically applied by farmers due to limitations in water resources in semiarid areas during summer and overlapping with high water demand by other annual crops (Vafabakhsh et al., 2010). In an experiment conducted using a lysimeter in Mashhad (36 N, 59 E, 985 msl) followed by an on-farm investigation in Torbat-e-Hydarieh (35 N, 59 E, 1333 msl), four summer irrigation treatments (one irritation in July, one irrigation in August, two irrigations: one in July and another in August, and no irrigation) were investigated on saffron yield. The results of the lysimeter showed that August irrigation increased saffron yield, while July irrigation had no effect and the two irrigations in July and August decreased the yield. On-farm research showed that summer irrigations had

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SECTION | II Safron production

no significant effect on saffron yield but resulted in earlier emergence of flowers after the first autumnal irrigation (locally known as Basar-Ab) (Mosaferi-Ziaoddini et al., 2007). Summer irrigation at the flower differentiation stage has a positive effect on saffron flowering, and it was reported that the stigma yield in irrigated and no summer irrigated fields was 4.9 and 3.39 kg ha21, respectively (Behdani et al., 2015). While under Afghanistan climatic conditions summer irrigation has some positive impacts on saffron flowering, it is not recommended due to the high risk of corm fungal infection from waterlogging (Katawazy, 2013). Behdani et al. (2008) in a study at agroecosystem scale in a dry region of Iran concluded that the mean yield of saffron on farms with summer irrigation application was higher than on farms with nonsummer irrigation management (4.9 vs 3.35 kg ha21). Summer irrigation on 10 July, which matches with initiation and differentiation of vegetative organs in corm buds, is harmful because increasing the soil moisture at this time prolongs the vegetative phase and disrupts the reproductive stage. However, irrigation later than 10 August, which coincides with initiation and differentiation of reproductive organs, encourages reproductive growth and increases saffron flowering (Hosseini and Mollafilabi, 2016). Summer irrigation can also affect the date of preflowering irrigation in autumn. When summer irrigation is applied, the time of preflowering irrigation ranges from the 10 to 30 of October (depending on the climatic conditions); otherwise, around 10 November is an appropriate time for the first autumnal irrigation (Behdani and Fallahi, 2015).

6.4

Rainfed saffron production

Frequent irrigation is not necessary in saffron production, due to its low water demand, which can be satisfied by the scarce rainfall when it is grown in semiarid regions. Even in Mediterranean environments, saffron is not watered in many cultivated areas (Gresta et al., 2008). For example, in a study in Balazote, Spain (38 N, 02 W, 740 masl), which has a continental Mediterranean climate, no irrigation was applied due to sufficient rainfall (248.5 mm) from November to April (Lopez-Corcoles et al., 2015). In Navelli, Italy, rainfed saffron production is also a common practice (Tammaro, 1999). Saffron is a drought-tolerant plant, and thus its rainfed production is possible if the amount and distribution of rainfall is favorable. For example, in central Spain the annual rainfall is 300
500 mm, and irrigation is generally not required. The timing and amount of rainfall during saffron vegetative growth, in addition to the interactions between temperature and humidity, are two critical factors for successful flowering. However, high moisture can encourage the spread of parasitic fungi in the corms (Halvorson, 2008). In Kashmir Valley, India, saffron fields are watered naturally with the rainfall. In this region, farmers rely only on rain, resulting in lower yield when it is not sufficient. The amount of rainfall in Srinagar, Kashmir, is about 400 mm during the saffron growing season, but with an irregular distribution, which usually causes some water stress (Kafi and Showket, 2007). Under rainfed saffron production, a preflowering irrigation at mid-to-end of October results in flower initiation similar to what occurs in irrigated plants. Accordingly, for rainfed conditions, a preflowering irrigation should be done when water is accessible (Sepaskhah and Kamgar-Haghighi, 2009). In a 6-year study, saffron corm and stigma yield under Khorramabad climatic conditions (33 N, 48 E, and a long-term annual rainfall of 520 mm, 1150 m elevation) were studied under rainfed and irrigated systems. With rainfed treatment, crops relied just on rainfall (about 370 mm during saffron growth season), while in the irrigated system irrigation was applied before the flowering stage in October. There was no significant difference between irrigated and rainfed production in terms of stigma yield, with an average yield of 7.5 and 7.1 kg ha21 during the 6 years of the study, respectively. The results also showed that there was not a considerable difference between the two production systems in terms of corm production, which was 23.9 and 22.9 tons ha21 under irrigated and rainfed conditions, respectively (Khademi et al., 2014). Juan et al. (2009) in a study in Albacete, Spain with annual rainfall of 249
586 mm during 4 years of study concluded that rainfed saffron production is possible, although 80 mm irrigation increases yield (Fig. 6.7).

6.5

Factors affecting water requirements

Saffron water requirements are affected considerably by climatic conditions and thus climate change is a main factor affecting crop water demand. Almost from the beginning of the twenty-first century, along with the change in climate parameters especially in arid and semiarid regions such as Iran, saffron has been exposed to some unfavorable climatic conditions including extreme temperatures and low rainfall during its growth cycle, led to yield reduction. Moreover, the local extinction of this species has started in southern regions of Khorasan province, and it has been predicted that saffron geographical distribution in the province will change from a spread to an island pattern in the coming years (Behdani and Fallahi, 2015; Fallahi et al., 2018). A modeling study under Khorasan province conditions revealed that

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FIGURE 6.7 (A) Number of flowers per square meter and (B) fresh weight of stigma (g m22) under irrigated and rainfed saffron production systems. Means followed by the different letters have significant difference at the 5% level of probability. From Juan, J.A., Corcoles, H.L., Munoz, R., Picornell, M., 2009. Yield and yield components of saffron under different cropping systems. Ind. Crops Prod. 30, 212
219.

saffron flowering will be delayed from current period (mid-November) to early-to-late December by the 1.5 C
2 C increase in air temperature due to climate change (Koocheki et al., 2009). In another study under controlled conditions for evaluation of the effect of increase in air temperature due to climate change, it was concluded that flowering failed when corms were kept at a temperature of 30 C during summer for 70, 90, or 120 days (Koocheki et al., 2010). It also has been foreseen that saffron water requirements will increase during the following period due to increased evapotranspiration and reduction in rainfall (Behdani and Fallahi, 2015; Fallahi et al., 2018). Results of a study in Southern Khorasan province revealed that under climatic change conditions, saffron water requirements rose continuously during the study period (2014
40). The average increase in water requirements was 62 mm. The highest and the lowest requirement were in Birjand (95 mm) and Qaen (33 N, 59 E, 1440 msl) (40 mm) counties, respectively. The mean water requirement of saffron is expected to increase from 425 mm in 2014 to 487 mm in 2040 (Jafarzadeh et al., 2015). A study in Kashmir, India, also showed that reduction in rainfall due to climate change is a strong driving factor for the reduced yield of saffron (Eajaz et al., 2018). The visible effects of climate change on rainfed saffron production systems in Kashmir were seen from 1999. Before this year, there was an annual rainfall of 1000
1200 mm with a good distribution during the growth season, but these days the precipitation is about 600
800 mm. Usually the critical months of preflowering (September and October) are dry. with inadequate rainfall, sprouting and flowering is delayed. Accordingly, the day and night temperature requirements for proper flowering are not met along with moisture availability in soil and thus the yield is reduced. Therefore artificial sprinkler irrigation during the preflowering stage is required to mitigate the impact of climate change (Nehvi et al., 2010). Finding target candidate genes, which can improve drought resistance and application of modern genetic modification techniques to introduce inbuilt tolerance to saffron plants will also mitigate the effect of climate change on this plant (Husaini, 2014). It has been shown that rainfall during March and April strongly affects the corm weight of saffron, which is a key factor affecting saffron yield in the following year (Vafabakhsh et al., 2010). Research conducted in dry regions of Southern Khorasan province, Iran, showed a positive correlation between precipitation and air humidity with saffron growth and yield (Kouzegaran et al., 2014). Saffron water requirements are also heavily dependent on evapotranspiration, which has a close relationship with latitude and altitude. In the highlands with low evapotranspiration, lower irrigation is needed (Fallah Ghalhary and Ahmadi, 2015). The field age is another factor that affects saffron water demands, and water consumption increases with the field age due to more corm density and leaf area resulting in higher transpiration (Behdani and Fallahi, 2015).

6.6

Irrigation methods

Applying an appropriate irrigation method promotes crop yield and water-use efficiency. In saffron production, the basin and furrow methods are the most common methods of irrigation, with few research findings on new irrigation methods such as sprinkler, drip, and subsurface. Azizi-Zohan et al. (2006) found that the total number of corms and number of small corms per m2 (,4 g) with the furrow irrigation method was significantly higher than with basin (flood) irrigation, whereas the total yield of corms and the number and weight of large corms ( . 8 g) were higher in basin than in furrow irrigation. Accordingly, flowering was improved significantly when basin irrigation was practiced (Table 6.14). The corms are not deep enough in the soil for furrow irrigation. Therefore cold stress in winter and heat in summer may impose undesirable effects on the vegetative and reproductive growth of saffron. Furthermore, water

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TABLE 6.14 Corm and stigma production of saffron in two different irrigation methods. Irrigation method

Stigma yield (kg ha21)

Forage yield (kg ha21)

No. of large corm ( . 8 g) per m2

Corm yield (tons ha21)

First growing season

Second growing season

Basin irrigation

1.98a

3.82a

604a

52.1a

10.8a

Furrow irrigation

0.44b

0.84b

421b

17.4b

6.5b

In each column, means with at least one similar letter are not significantly different at 5% probability level. Source: Data from Azizi-Zohan, A.A., Kamgar-Haghighi, A.A., Sepaskhah, A.R., 2006. Effect of irrigation method and frequency on corm and saffron production (Crocus sativus L.). J. Water Soil Sci. 10(1), 45
54 (in Persian).

stress is more common when corm planting depth is not enough due to faster depletion of soil surface water (Sepaskhah and Kamgar-Haghighi, 2009). In a 2-year study, using the basin irrigation method, saffron yield was 2.32 and 5.4 kg ha21, while the yield were only 0.53 and 1.20 kg ha21under furrow irrigation in the first and the second years, respectively. Hence, water-use efficiency was significantly higher in basin irrigation (Azizi-Zohan et al., 2009). However, the place in which corms are planted, inside or out of the furrow, is important for furrow irrigation. For example, the results of Yarami and Sepaskhah (2015) on brackish water irrigation revealed that with the in-furrow planting method the saffron yield was 3.5 times higher than with basin planting. This observation was attributed to appropriate soil temperature for corm growth with the in-furrow planting. In addition, stigma qualitative indices including picrocrocin and crocin concentration for the in-furrow planting were higher than with the basin method by about 8% and 4%, respectively (Yarami and Sepaskhah, 2016). Evaluation of saffron corm production with basin and furrow irrigation methods over 9 years showed that the number of replacement corms in both irrigation methods increased up to the 6 growing season and then decreased. In most studied years, the corm production with furrow irrigation was higher than with basin irrigation. Replacement corm yield up to the fourth growing season was higher with basin irrigation, but after that furrow irrigation showed better results. Corm yield showed a decreasing trend from the third growing season with basin irrigation and from the fifth growing season with furrow irrigation (Khazaei et al., 2013). In a study in Jolgeh
Rokh, Iran (a region in Torbat-e-Hydarieh; 35 N, 58 E, 1770 msl) the effect of irrigation levels (100%, 75%, and 50% water requirement) and methods (basin, drip, and sprinkler irrigation) was studied on leaf and replacement corm growth of saffron in a 4-year-old field. The saffron water requirement in the studied area was calculated as 2730 m3 ha21 by the Penman Monteith method. Reducing the irrigation level from a 100% to a 50% crop water requirement reduced the number of replacement corms and their weight. Among irrigation methods, the best treatment in terms of underground and aboveground growth criteria was drip irrigation, followed by the sprinkler method. For example, in basin, sprinkler, and drip irrigation methods, the leaf dry weight were 264, 369, and 416 g m22, number of replacement corms per m2 were 545, 664, and 731, replacement corm diameter were 22.2, 22.7, and 24.4 mm and corm dry weight were 1020, 1119, and 1366 g m22, respectively (Karimiferezgh et al., 2018). Under Morocco conditions, the drip irrigation method can increase saffron flowering while reducing water use, but it is not widely applied due to setup costs (Filipski et al., 2017). In Iran, flood irrigation (basin method) is more common between traditional farmers, but pioneer farmers also employ new irrigation methods such as sprinkler and drip irrigation. The artificial rain is useful in heavy soil to avoid root suffocation. It also distributes water uniformly over the field and enables foliar application of fertilizers during the last phase of the growing season (March
April) when the saffron root system is weak in nutrient absorption. Fertigation (use of fertilizers in irrigation water) is also possible in drip irrigation of saffron fields (Behdani and Fallahi, 2015; Behdani et al., 2018).

6.7

Water quality

6.7.1 Water quality parameters Water quality refers to the physical, chemical, and biological features of water. The best growth and yield of saffron is obtained when the concentration of salt-induced ions, especially sodium, in irrigation water is low. Water electrical

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83

conductivity (EC) above 2.9 dS m21 has a severe reduction effect on saffron growth and flowering (Behdani et al., 2017). In a research study, an increase in irrigation water pH from 7.4 up to 8.5 had an increasing effect on saffron stigma yield especially in newly established fields. In contrast, an increase in sodium absorption ratio, total dissolved solids, bicarbonate, sodium, sulfate, and magnesium in irrigation water exerted a negative effect on saffron yield in all 3- to 7-year-old fields, while carbonate increased stigma yield (Nasiri Khorasani, 2011). The biological features of irrigation water are also highly important especially in organic fields of saffron. Behdani et al. (2017) reported that application of sewage water in saffron organic fields and their vicinity is forbidden. They noted that all necessary tools such as fencing around water supplies and filtration should be applied to reduce the microbial load of water. Hence, microbial contamination of the ground and surface water resources should be tested yearly and every 3 months, respectively, to ensure the supply of healthy water for saffron irrigation.

6.7.2 Salt stress in saffron In the saffron cultivation areas of Iran, the concentration of some ions, especially salt-induced ions in irrigation water, is relatively high. It has been reported that the use of irrigation water with a salinity level of 1.7 dS m21 can reduce saffron flower yield by 49%, while no flowers are produced at salinity level of 4.0 dS m21, although leaf growth occurs. Furthermore, 50% flower yield reduction occurred at soil water salinity of 3.6 dS m21. The EC threshold of the irrigation water was estimated to be 0.13 and 0.48 dS m21 for flowers and roots, respectively (Sepaskhah and Yarami, 2009). The results of another study showed that saffron yield declined by 38% in response to increasing water salinity from 0.45 to 3 dS m21. Therefore it was concluded that saffron is a salt-sensitive crop, but its high salt sensitivity could be mitigated by cow manure application and the in-furrow planting method (Yarami and Sepaskhah, 2015). Nasiri Khorasani (2011) reported that increase in water EC has a negative impact on saffron flowering, where stigma yield was about 5.2 and 4.3 kg ha21, in 0.5 and 1.5 dS m21, respectively. In another study, it was reported that the saffroncoloring strength (crocin), bitterness (picrocrocin), and aromatic strength (safranal) decreased by 9%, 13%, and 18%, respectively, at a salinity level of 3 dS m21 compared with the control treatment (Yarami and Sepaskhah, 2016). Mzabri et al. (2017b) also observed a reduction in flowering and vegetative growth of saffron, when the concentration of NaCl in irrigation water increased from 1 to 3 g L21. Saffron is very sensitive to salinity, and its production function for flowering has an irrigation water salinity threshold of 0.13 dS m21 and line slope of 28.3% per unit of increase in water salinity, under pot condition without winter precipitation. Irrigation water salinity of 1.7 dS m21 or soil water salinity of 3.4 dS m21 reduces flower production by 50%. So can brackish water be used for its production? In areas with sufficient winter rainfall (300
400 mm) it is possible to use brackish water if the corms are planted by the in-furrow method alongside the application of 30
60 tons ha21 cow manure. In this situation, the salinity threshold for soil saturation extract and soil saturation salinity for 50% yield reduction reaches 1.1 and 2.3 dS m21, respectively. In addition, with the in-furrow planting method the flowering with irrigation water salinity of 0.45 and 3 dS m21 increases by two- and fourfold, respectively, compared with basin planting. Moreover, in-furrow planting and cow manure application reduce the negative effect of brackish water on stigma quality (Sepaskhah, 2018). The highest yield of saffron was obtained in the second growing season instead of the third growth year under application of brackish water (445 mm rainfall 1 263 mm irrigation water), probably due to salt accumulation. Water-use efficiency in the second growth season reduced when irrigation water salinity was higher than 2 dS m21. It reached from 4 g m23 in 1 dS m21 to 2.5 g m23 in water salinity of 3 dS m21. However, cow manure application and the infurrow planting method (by threefold) increased the water-use efficiency. But the leaching fraction must be considered to control the soil salinity from brackish water. In addition, continuous planting of saffron in the same field causes an accumulation in ions and toxic and allelopathic substances. To solve this problem, leaching during cultivation years with double volume of soil porosity (B360 mm), in addition to 400 mm annual precipitation, can be useful (Sepaskhah, 2018).

6.8

Water-use efficiency and productivity

Water availability is increasingly restricted and is expected to become a serious problem in the future. Around one-third of the world’s surface is covered by arid and semiarid areas facing water shortage. From the beginning of the twentyfirst century, given the world’s growing need for food, exploitation of lands under abiotic stresses is considered. Therefore, the use of plants that are more resistant to environmental stresses has a high importance (Fallahi et al., 2015c). Saffron is one of the most efficient plants in terms of water consumption (Fallahi et al., 2014). The results of

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calculation of water-use efficiency in saffron and wheat production systems in the Qaen region (located in south Khorasan province, Iran) showed that water-use efficiency based on total aboveground biomass and grain yield was 0.84 and 0.34 kg m23 for wheat and 0.36 and 0.002 kg m23, based on total biomass and stigma yield, for saffron, respectively. Economic water-use efficiency (water productivity) was estimated to be 23,706 and 1837 Rials (  0.73 and 0.06 US dollar in 2016) per cubic meter of water in saffron and wheat production systems, respectively. There was a significant difference of the economic water-use efficiency for different ages of saffron fields and its maximum value was obtained for 5-year-old fields (Yaghoubi et al., 2016). Considering the increasing trend of drought stress in Iran and the fact that saffron is a water-efficient crop, its production is becoming more common in new areas, in particular higher latitudes, like Golestan province (36
38 N, 54
57 E) in the northern regions of Iran. In a study on the causes of expansion of saffron cultivation in the temperate mountain areas of Golestan, it was shown that 82% of farmers believe that saffron is more climatically resistant compared with other local crops. Saffron economic income per hectare (92,372,000 Rials; 1$ was equal to 34000 Rials, in 2015) was also 15 and 8.7 folds of potatoes and cereals, respectively (Adeli and Anabestani, 2015). The results of Shamsabadi et al. (2016) also revealed that the physical productivity of water for saffron in Torbate-Jam (35 N, 61 E, 928 msl) and Bakharz (35 N, 60 E, 1279 msl) counties is 1.07 and 1.84 g m3 of water, and the economic productivity of water is 56,428 and 100,970 Rials per cubic meter (1$ to 36,000 Rials, in the relevant year), respectively. This difference between the two cities was attributed to better quality of water and soil as well as older history of saffron production and hence more experienced farmers.

6.9

Physiological responses of saffron to water stress

Water stress adversely affects plant growth, development, productivity, and other physiological aspects of plants. Under drought stress, plants adopt some biochemical and physiological approaches to acclimate with water shortage conditions (Behdani and Fallahi, 2015). There is a high correlation (r 5 0.92**) between the soil water potential and the photosynthetic rate of saffron (Renau-Morata et al., 2012). Lower soil water potentials leads to lower relative water content in leaves, mother corms, daughter corms and roots of saffron and also lowers photosynthetic rate (AN) (Fig. 6.8A), reductions in the substomatal CO2 concentration and the rate of electron transport, but also cause an increase in proline content and water-use efficiency (Fig. 6.8B). However, there is no dramatic photosynthetic rate reduction under very low soil water potential. The water supply from the corms and roots to the leaves could alleviate the negative effects of low soil water potential on the saffron leaves during a particular stage of plant growth (Renau-Morata et al., 2012).

FIGURE 6.8 Relationship between (A) photosynthetic rate (AN) and (B) water-use efficiency of saffron (WUE) with soil water potential under field condition. From Renau-Morata, B., Nebauer, S.G., Snchez, M., Molina, R.V., 2012. Effect of corm size, water stress and cultivation conditions on photosynthesis and biomass partitioning during the vegetative growth of saffron (Crocus sativus L.). Ind. Crops Prod. 39, 40
46.

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TABLE 6.15 Effect of different levels of drought stress on the yield and biochemical parameters of saffron. Treatment (% ETo)

Total chlorophyll content (mg g FW)

Quantum yield of PSII

Leaf proline content (μg g21 FM)

Leaf soluble sugars content (μg g21 FM)

Malondialdehyde content (nmol g21 FM)

100

0.91a

0.75a

201b

2241b

0.00134a

60

0.83a

0.72a

361b

2881ab

0.00144a

40

0.71

a

a

a

Treatment (% ETo)

Total phenols content (µg g FM)

Relative water content (%)

Leaf water potential (MPa)

Corm weight (g per plot)

Stigma yield (g per plot)

100

296.2b

74a

2 7.6b

412.4a

0.37a

60

374.5ab

69a

2 8.3b

359.3a

0.34a

a

2 10.6

a

0.26a

40

452.6

0.70

a

65

708

3035

a

a

367.8

0.00176a

In each column, means with at least one similar letter are not significantly different at 5% probability level.

Source: Data from Mzabri, I., Legsayer, M., Aliyat, F., Maldani, M., Kouddane, N.E., Boukroute, A., Bekkouch, I., Berrichi, A., 2017a. Effect of drought stress on the growth and development of saffron (Crocus sativus. L) in eastern Morocco. Atlas J. Biol. 17, 364
370.

The results of the physiological response of saffron to two levels of water availability (100% field capacity and full drought) under greenhouse conditions showed that both chlorophyll a and b content decreased significantly under drought stress. The relative water content also decreased in drought treatment and the use of large mother corms for saffron cultivation proposed as a guarantee for further growth and survival of saffron under drought stress conditions (Sabet Teimouri et al., 2010). It was reported that an increase in qualitative factors including picrocrocin, crocin, and safranal could be obtained by intensifying moisture stress (Hosseini and Rahimi, 2016). Koocheki and Seyyedi (2016) obtained similar results where crocin and picrocrocin content increased in response to decreasing irrigation levels. Mzabri et al. (2017a) in a study conducted on a 4-year-old saffron plantation in Oujda, Morocco tested the physiological response of saffron to three water regimes including receiving 100%, 60% (moderate water deficit), and 40% ETo (severe water deficit). Drought stress decreased the chlorophyll content and also resulted in a slight decrease in the PSII quantum yield. In addition, proline, soluble sugars, and total phenols accumulated, which resulted in keeping the relative water content and the malondialdehyde content as high as nonstressed plants. Overall, the morphophysiological adaptation occurred even at severe water deficit, which resulted in a low stigma yield decrease (Table 6.15). In a follow-up to the experiment, Mzabri et al. (2017c) tested the vegetative and reproductive growth of replacement corms, which were produced under drought (100%, 60%, and 40% ETo) or salt (0, 1, 3, and 5 g L21) stresses. Their results showed that there was no significant difference between corms obtained from stressed plants with those obtained from nonstressed plants in terms of flowering and vegetative growth parameters.

6.10

Beneficial water-related approaches for saffron production

Low water availability is the most important limiting factor for crop production in dry regions. Therefore it is necessary to adopt suitable approaches to increase plant tolerance to drought stress. Application of soil conditioners like superabsorbents, zeolite, and organic manures is a suitable strategy for sustainable saffron production in areas affected by drought stress (Behdani and Fallahi, 2015; Fallahi et al., 2013a). In a pot experiment the percentage and rate (rapidity) of sprouting of saffron in response to application of calcic, potassium, and soil zeolites in two rates (20 and 60 g kg21) plus a control treatment was studied and it was concluded that using K-zeolite, especially at the rate of 60 g kg21, can increase the emergence percentage and decrease the mean time required for saffron corm sprouting (Ahmadee et al., 2014). In another pot experiment, the combined effect of K-zeolite levels (0%, 0.5%, 1%, and 2%) and water application (traditionally three times irrigation levels: one before flowering and one after flowering and also one at the end of growing season, deficit irrigation at 70% soil moisture depletion, and full irrigation to keep soil moisture at field capacity point) was studied. Stigma dry weight increased by using more zeolite and irrigation water, and the maximum yield was obtained at full irrigation along with 1% or 2% zeolite, followed by deficit irrigation plus 2% zeolite. In all irrigation levels, the saffron stigma, style, and petal dry yields increased in the presence of zeolite

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(Khashei-Siuki et al., 2016a). In addition, the effects of the same levels of irrigation and zeolite revealed that applying zeolite in all irrigation treatments increased the corm weight and number of large corms and decreased the production of small corms (Khashei-Siuki et al., 2016b). Since saffron fields in arid and semiarid climatic conditions have relatively low moisture content, superabsorbent polymers (SAPs) might increase growth and yield of saffron (Khorramdel et al., 2014). SAPs are hydrophilic crosslinked polymers that can absorb water to act as a water reservoir near the roots. These polymers grow into several times their original size, after absorbing water. SAPs delay the time to wilting and prolong plant survival under drought stress by increasing the water-holding capacity and nutrient retention of soil. Therefore they are helpful materials to increase crop production in arid and semiarid areas (Samadzadeh et al., 2016). However, water quality has a significant effect on SAP effectiveness, therefore, salinity greatly reduces the water-holding capacity of SAPs (Fallahi et al., 2015a). In one study the effect of different levels of SAPs (0, 0.1, 0.2, 0.4, and 0.8 wt.% based on dry weight of soil) showed that with an increase in SAP rate the flowering rate, corm yield, number of flower, flower yield, and stigma dry yield significantly increased (Khorramdel et al., 2014). In a similar study, application of 40 kg ha21 SAP in a saffron field increased the number of replacement corms by 13%, total weight of replacement corms per plant by 36%, mean weight of replacement corms by 29%, and mean number of buds per corm by 27%, compared with the control treatment. Moreover, SAP application decreased the amount of nonstandard (,8 g) corms, while significantly increasing the production of large corms. In addition, the water-use efficiency in SAP application treatments (6.1 kg large corm m23) was more than the control (4.34 kg m23) treatment (Fallahi et al., 2016a). In another study the combined effects of irrigation intervals (2, 3 and 4 weeks applied from 5th March up to the end of growing season; equals to 3000, 2400, and 1800 m3 ha-1, respectively), super absorbent, humic acid and mycorrhizal symbiosis was studied. The results showed that application of superabsorbent under irrigation every 4 weeks could compensate for the lack of water and increase the flower yield of saffron. Application of superabsorbent and humic acid enhanced the number of buds, number of replacement corms, and mean weight of replacement corms. The maximum corm and stigma yields were also obtained in those plants that received superabsorbent (Table 6.16) and were inoculated with mycorrhiza. In addition, water-use efficiency was improved partially when superabsorbent humic acid and mycorrhiza were used (Table 6.17) (Aghhavani-Shajari, 2017). In previous studies, the positive effect of a humic substance on mitigating the effects of drought stress have been reported, and it has been shown that this organic resource has increased the corm and flower production of saffron in dry regions (Fallahi et al., 2016b; Koocheki et al., 2016c, 2012a,b). Mycorrhizal inoculation also facilitates water and nutrient uptake by saffron under stressful conditions. The presence of vesicular arbuscular mycorrhiza, particularly Acaulospora morrowiae and Glomus coronatum, has been observed in saffron fields (Zare Maivan and Nakhaei, 2000). It has been well documented that saffron has good symbiosis with Glomus species and the inoculation improves its chlorophyll content and vegetative growth parameters such as leaf, root, and corm weights (Shuab et al., 2016). Foliar application of some growth regulators may also encourage better saffron growth and development in dry regions. For example, Maleki et al. (2011) observed that abscisic acid (ABA) spraying enhanced saffron drought tolerance as shown by higher relative water content and more activity of antioxidant enzymes. Besides these options, the use of modern irrigation methods in saffron production may also increase its water-use efficiency (Behdani and Fallahi, 2015). Cultivation under controlled environments may also be a useful strategy for saffron production in areas affected by water shortage (Mollafilabi et al., 2014). In this cultivation method, the growth environment and nutrition of the plants TABLE 6.16 Effect of natural superabsorbent on saffron performance in Mashhad. Natural superabsorbent* (kg ha21)

Flower yield (kg ha21) 2014 a

Dry stigma yield (kg ha21)

2015 b

2014

2015

a

b

Corm yield (g m22) 2014

Mean replacement corm weight (g)

2015 a

a

2014

2015

a

0

91.5

226.7

0.71

2.66

2354

3028

3.13

2.60b

500

96.0a

384.0a

1.17a

4.35a

1531b

3443a

2.01b

3.39a

In each column, means with at least one similar letter are not significantly different at 5% probability level. *Zeolite: 12.5% 1 perlite: 12.5%, cotton powder: 12.5% 1 sawdust: 12.5% 1 cow manure: 25%1 pit: 25%. Source: From Aghhavani-Shajari, M., 2017. Study the Possibility of Increasing Saffron (Crocus sativus L.) Corm Size and Flower Yield Through the Farming Managements (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

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TABLE 6.17 Water-use efficiency of saffron as affected by natural superabsorbent, humic acid application, and mycorrhizal inoculation using Glomus intraradices. Treatments

Water-use efficiency based on stigma (g m23)

Water-use efficiency based on corm (kg m23)

2014

2015

2014

2015

0.147a

0.794b

6.57a

8.06a

0.280a

1.285a

5.06a

8.63a

3000

0.265a

0.792c

5.79b

8.42ab

2400

0.157b

1.372a

4.69c

7.71b

1800

0.249a

0.954b

6.98a

8.91a

0.190a

1.104a

4.60c

7.87b

0.206a

0.836b

5.83b

8.41ab

a

a

a

21

Natural superabsorbent (kg ha ) 0 500 3

21

Water application (m ha )

Fertilization by natural resources Control Mycorrhiza (1.5 kg m22*) 21

Humic acid (15 kg ha )

0.275

1.278

7.03

8.77a

In each column, means with at least one similar letter are not significantly different at 5% probability level. *15 g of mycorrhiza (spores plus companion matter) for each planted corm. Number of alive spores 5 120
150 per g. Source: From Aghhavani-Shajari, M., 2017. Study the Possibility of Increasing Saffron (Crocus sativus L.) Corm Size and Flower Yield Through Farming Management (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

can be carefully controlled, resulting in higher yield and generally higher quality (Maggio et al., 2006; Fallahi et al., 2017b). In addition, under controlled culture, lower water is needed for plant production, which is an advantage in arid regions. For more details, see Chapter 12: Emerging innovation in saffron production. A vertical culture system likewise may increase the resource-use efficiency, especially water, the main limiting factor for crop production in drylands (Ali-Ahmad et al., 2017). Examples of this production system include the use of vertical columns, vertically suspended grow bags, and plant factory approaches (Touliatos et al., 2016). This method for saffron production may be used in controlled or outdoor environments. It appears that vertical production in an outdoor environment mainly can be used to produce saffron on a household scale and to meet family needs.

6.11

Saffron response to flooding

The occurrence of flooding is a rare phenomenon in many dry regions of saffron production. However, some heavy rainfalls, especially in clay soils, during its vegetative growth can cause flooding stress, which is harmful for plant growth. Moreover, in some regions of saffron distribution there is annual rainfall up to around 1000 mm, with higher risk of waterlogging. In one study, the effect of silver ion (as an ethylene inhibitor) was studied on the growth of saffron under flooding conditions. Flooding for 10 days reduced the growth of roots and leaves, but corms soaked with 40 or 80 ppm concentration of nanosilver alleviated the effect of flooding stress on the plant growth (Rezvani et al., 2012). In similar research, it was reported that flooding decreased plant dry weight and corm production but had no significant effect on root growth. Under waterlogging conditions, foliar application of nanosilver (50 or 100 ppm) caused an increase in plant height and corm number (Sorooshzadeh et al., 2012).

6.12

Indigenous irrigation knowledge

Local knowledge is based on experiences farmers have gained over the years. This knowledge has been tested over centuries and is adapted to the local environment and culture (Fallahi et al., 2013a,b). In a study on indigenous knowledge of saffron production in Southern Khorasan province, it was shown that most farmers (77%) believe that preirrigation before planting and then corm sowing when the soil moisture is appropriate for planting operations (wet-planting)

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SECTION | II Safron production

promotes saffron growth and yield. In addition, most of them disagreed with irrigation before corm lifting from old fields (98.5%) and also immediately after corm sowing in new fields (100%). About 90% of saffron farmers believe that in a year with normal rainfall (120 mm), four or five irrigations are sufficient for this plant, while only 3% believe more than seven irrigations during the saffron growth season are needed. Furthermore, 20% of farmers note that if freezing occurs, winter irrigation has a negative effect on saffron flowering. All farmers had a negative opinion on summer irrigation due to the risk of corm decay in high temperatures in summer. They reported that the last irrigation in mid-April (locally referred to as Zard-AB) has a positive effect on corm growth. Finally, 80% of farmers disagreed with application of floodwater in saffron fields due to decreasing of soil aeration (Fallahi et al., 2015b). In another study in Southern Khorasan province, the indigenous knowledge of saffron irrigation was compared with the results obtained from scientific research. The aim was to determine what, for each agronomic operation, percentage of the farmers operates in accordance with the findings of scientific research. The finding revealed that 73% of farmers were aware of proper timing of irrigation. The correct answers were 35% for suitable time of the first irrigation, 33% for suitable irrigation for making larger corm, 31% for number of days after planting for irrigating, 28% for the most important irrigation in saffron production, and 24% for suitable number of irrigation between reproductive and leaf senescence stages (Khozeymehnezhad et al., 2016). Evaluation of local saffron producers in Torbat-e-Hydarieh in Iran also revealed that the majority of farmers (55%) use four irrigation rounds during winter and spring. Some farmers had a negative (48%), some positive (34%), and others neutral opinion about summer irrigation. Finally, the majority of farmers (70%) believed that 1
20 August is a suitable period for application of summer irrigation (Aghhavani-Shajari et al., 2014).

6.13

Conclusion

Saffron is one of the most common crops in dry regions where most of its water requirement is provided from rainfall. Rainfed production is a common method where rainfall is about 400
500 mm, with good distribution during vegetative growth. However, irrigation is needed in dry and semidry areas to gain the desired yield. Therefore application of approximately 3500 m3 ha21 during the growth cycle can meet water requirements in these areas. This volume of water can be applied six times, two before and after the flowering period and four during the vegetative growth, especially when the interval between two useful consecutive rainfalls is more than a month. It is recommended that two irrigations be applied at the end of growth, which lines up with the rapid growth of replacement corms. In addition, saffron water requirements are lower in the first growing season and then an up-trend occurs during the coming years. Irrigation before corm lifting in old fields (for application of lifted corms to establish new fields) and immediately after corm planting in new established fields is not recommended for saffron. This means the first irrigation to stimulate the emergence of flowers must be postponed to autumn, when saffron corms are planted during spring or summer. Late and early preflowering irrigation has a negative impact on saffron growth and yield. The date of preflowering irrigation is strongly dependent on climatic factors especially temperature, and the best time of this irrigation varies, even in specific regions. Summer irrigation during flower differentiation may be a useful practice, but it must be avoided if there is a risk of corm disease. Basin and furrow irrigation are the most common methods of irrigation and adaptation of new methods like drip and sprinkler irrigation will be needed. It appears that providing 75% crop water is a good strategy in areas with a low water accessibility. In these regions, application of water-holder materials such as superabsorbents, zeolite, and organic manures and the use of symbiotic microorganisms like mycorrhizal fungi are useful ways for more efficient use of water. Saffron production under controlled environments may also increase water-use efficiency.

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555. Behdani, M.A., Fallahi, H.R., 2015. Saffron: Technical Knowledge Based on Research Approaches. University of Birjand Press, Birjand (in Persian). Behdani, M.A., Nassiri-Mahallati, M., Koocheki, A., 2008. Evaluation of irrigation management of saffron at agroecosystem scale in dry region of Iran. Asian J. Plant Sci. 7, 22
25. Behdani, M.A., Fallahi, H.R., Aghhavani-Shajari, M., 2015. Future Crops. University of Birjand Press, Birjand (in Persian). Behdani, M.A., Jami Al-Ahmadi, M., Fallahi, H.R., 2016. Biomass partitioning during the life cycle of saffron (Crocus sativus L.) using regression models. J. Crop Sci. Biotech. 19 (1), 71
76. Behdani, M.A., Jami Al-Ahmadi, M., Mahdavi-Damghani, A., Fallahi, H.R., 2017. Standardization and Codification of Technical Knowledge of Iranian Organic Saffron. University of Birjand Research Project, Iran (in Persian). Behdani, M.A., Fallahi, H.R., Sardar, M., 2018. Technical Knowledge of Saffron Production. Haft-Rang Press, Birjand (in Persian). Coskun, M., Gok, M., Coskun, S., 2017. Climate characteristics of karabuk and saffron cultivation. Int. J. Geograph. Geolog. 6 (3), 58
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Fallahi, H.R., Zamani, G.R., Aghhavani-Shajari, M., Samadzadeh, A., 2017b. Comparison of flowering and growth of saffron in natural and controlled culture systems. In: Proceedings of the Sixth National Congress on Medicinal Plants. May 9
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Saffron water requirements Chapter | 6

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Nehvi, F.A., Wani, S.A., Dar, S.A., Makhdoomi, M.I., Allie, B.A., Mir, Z.A., 2007. New emerging trends on production technology of saffron. Acta Hort. 739, 375
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Chapter 7

Saffron “seed”, the corm Alireza Koocheki and Seyyed-Mohammad Seyyedi Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 7.1 Introduction 93 7.2 Corm botanical criteria 93 7.2.1 Mother and daughter corms 93 7.2.2 Main and lateral buds 94 7.2.3 Root system 96 7.2.4 Developmental stages and phonological description 98 7.2.5 Corm nutrient content 98 7.2.6 Field age effect on corm production 101 7.3 Agronomical practices 101 7.3.1 Corm lifting time and storage 101

7.1

7.3.2 Planting time 7.3.3 Planting depth 7.3.4 Row spacing, corm density 7.3.5 Corm size/weight 7.3.6 Corm size classification 7.3.7 Planting beds 7.3.8 Application of hormones 7.4 Corm and climate change 7.5 Conclusion References

103 103 104 104 106 107 112 112 113 113

Introduction

Saffron (Crocus sativus L.) is globally known for its flowers value in food and pharmaceutical industries (Giaccio, 2004; Xi et al., 2007). Despite the numerous studies on saffron flowers, insufficient attention has been paid to saffron corms as the main important factor in saffron flowering. Although saffron’s phenological stages are essentially defined based on corm formation and growth (Gresta et al., 2008), agronomic management is performed mainly based on flower emergence rather than corm growth (Koocheki et al., 2016a; Rubio-Moraga et al., 2013). In 2016, Iran’s saffron cultivation area and production were 105,200 ha and 336 tons, respectively, with an average farm age of 6
10 years (Agricultural Statistics, 2017). Hence, 12.5% of the total farm area (13,150 ha) is estimated to be about 8 years old. On the other hand, given the average yield of 31.85 ton ha21 saffron corm from 8-year-old farms (Mollafilabi et al., 2015), the total annual harvest of saffron corm in Iran is recorded as 418,830 tons.

7.2

Corm botanical criteria

7.2.1 Mother and daughter corms Saffron is a sterile triploid geophyte plant, which is propagated by corms (Fernandez, 2004; Gresta et al., 2008). Botanically, corms are short and thick underground stems covered with fibrous reticulated leaf tunics to protect a creamy colored smooth epidermis (Molina et al., 2004b). The shape of saffron corm varies from circular to elliptical, and its base is usually flat or slightly curved (Kumar et al., 2009; Renau-Morata et al., 2013). The saffron plant is propagated by mother corms due to its sterility caused by being triploid (Nehvi et al., 2010). Mother corms, which are underground organs, have meristematic tissues that generate new corms as replacement or daughter corms (Fig. 7.1). Mother corms deteriorate gradually with increasing daughter corm growth. In other words, each mother corm produces new daughter corms before withering (Renau-Morata et al., 2012). Each daughter corm is also considered as a potential mother corm for the next growing season (Bhagyalakshmi, 1999). At the end of growing season, remaining mother corms (Fig. 7.2) appear as brown, oval, and flat disks attached to the daughter corms (Kumar et al., 2009). Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00007-1 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 7.1 Saffron above- and underground organs. (A) Stem sheath, (B) mother corm, and (C) absorbing (fibrous) roots. The formation and growth of saffron daughter corms affect flower yield during the next growing season. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

FIGURE 7.2 Daughter corm organography. (A) Remaining mother corm, (B) daughter corm, (C) fibrous reticulated leaf tunics, and (D) uncovered smooth epidermis corm. During each growing season, saffron propagates vegetatively by means of a tuberous-bulb formation and growth called the mother corm. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during the growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

Accordingly, saffron stigma yield highly depends on daughter corm growth in the previous growing season, because the reproductive mechanism at the onset of the growing season is affected by the concentration of nutrient reserves in the corms (Gresta et al., 2008; Koocheki and Seyyedi, 2019; Renau-Morata et al., 2012).

7.2.2 Main and lateral buds From a botanical point of view, growth induction and daughter corm formation is controlled by cell division in apical, subapical, and lateral (axillary) buds (Kumar et al., 2009). The apical and subapical buds are known as vegetativereproductive sprouts, while the lateral buds are differentiated for the production of leaves (Bhagyalakshmi, 1999; Kumar et al., 2009). By removing leaf tunics pale and circular lines become visible on the epidermis surface. In addition, the epidermis surface is creamy and yellowish in color and has dots on it. The brown apical and lateral buds are visible on the epidermis surface (Fig. 7.3). The apical bud is located at the tip of the corm, but lateral buds are found only on the sides (Abrishamchi, 2003; Kumar et al., 2009). Depending on mother corm size, the number of subapical and lateral buds range from 1 to 2 and from 2 to 10 buds, respectively (Koocheki and Seyyedi, 2015a; Molina et al., 2005a). Normally, buds become smaller

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FIGURE 7.3 Saffron corm morphology after removing leaf tunics. (A) Apical, (B) subapical, and (C) lateral (axillary) buds. From a botanical point of view, saffron reproduction is made through the growth of meristem tissues and establishment of new daughter corms produced by the mother corms. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

FIGURE 7.4 Saffron flowering due to stimulating reproductive buds on mother corms. (A) Stem sheath, (B) initial leaves, (C) petals, (D) stigma, (E) style, and (F) stamen. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

from the tip to the base of each corm (Tavakkoli et al., 2014). On the other hand, apical buds are more capable of forming daughter corms than lateral ones (Fig. 7.3). Saffron corm dormancy starts after drying aboveground parts, normally in May (Babaei et al., 2014; Koocheki et al., 2016b). The dormancy has two phases: true and apparent dormancy. Until mid-July, meristematic cells of the tip of the buds have detained activity, which is called true dormancy (Sadeghi et al., 2003). Apparent dormancy is also divided into two stages; during the first stages (until early August) vegetative organs start to develop (Abrishamchi, 2003), whereas in the second stage (until late August) reproductive organs differentiate (Koul and Farooq, 1982; Sadeghi et al., 2003). The dormancy is broken and flowering-related physiological processes start to occur at the end of August (Abrishamchi, 2003; Koocheki and Seyyedi, 2016a). The first flowers start to appear (Fig. 7.4) through increasing reproductive organ activity with decreasing temperature in autumn (Kumar et al., 2009; Rabani-Foroutagheh et al., 2013). Flowers appear before or after or at the same time of leaves appearance depending on weather conditions (Koocheki and Seyyedi, 2015a; Molina et al., 2004a). As can be seen from Fig. 7.5 a relative increase in air temperature, postpone flowering until the leaves have started to appear (Arsalani et al., 2015; Kumar et al., 2009). At this stage, daughter corm growth reduces, as more energy is needed for flowering and early growth (Maleki et al., 2011; Renau-Morata et al., 2012). After flowering, vegetative growth starts to increase and the first daughter corms appear on mother corms (Gholami et al., 2017; Koocheki et al., 2016a). The daughter corms may continue to form throughout the vegetative growth period (Fig. 7.6). However, the daughter corm growth rate reaches to its maximum in December (Koocheki and Seyyedi, 2015a; Renau-Morata et al., 2012). As mentioned earlier, apical buds are more capable of producing flowers and daughter corms in comparison with lateral buds (Amirshekari et al., 2007; Tavakkoli et al., 2014). However, buds stimulation increases competition between these buds (Sabet-Teimouri et al., 2010; Tavakkoli et al., 2014). Extensive use of water and fertilizers,

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FIGURE 7.5 Saffron flowering process in autumn. (A) Saffron flower appearance before its leaves emerge and (B) saffron flower appearance after its leaves emerge.

FIGURE 7.6 Daughter corm formation from mother corm. (A) A mother corm at planting time (11.93 g), (B) the mother corm at the end of the first growing season, and (C) daughter corms (average weight per corms: 1.71 g). From Koocheki, A., Seyyedi, S.M., 2016. Effects of corm size, organic fertilizers, Fe-EDTA and Zn-EDTA foliar application on nitrogen and phosphorus uptake of saffron (Crocus sativus L.) in a calcareous soil under greenhouse conditions. Not. Sci. Biol. 8, 461
467.

FIGURE 7.7 Formation of three large daughter corms (average weight: 10.31 g) from mother corm. 1 to 3: Dense and compact daughter corms at the end of the growing season. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

especially nitrogen as well as increased temperature, increase the competition between saffron buds (Chaji et al., 2013; Koocheki and Seyyedi, 2015b). To prevent competition, removing extra buds has been suggested (Kumar et al., 2009; Tavakkoli et al., 2014). Although removing weak buds may reduce daughter corm numbers, it may result in increased daughter corm weight, which plays a key role in increasing saffron yield (Fig. 7.7). In general, in corms weighing 6
8 g, the most desirable bud number is three: two lateral buds and one apical bud (Tavakkoli et al., 2014).

7.2.3 Root system There are two structurally and functionally different types of roots in saffron: absorbing (fibrous) and contractile roots (Fig. 7.8). The fibrous roots (about 1
3 mm diameter) that form from the base of each corm absorb water and nutrients

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FIGURE 7.8 Formation and growth of root system in saffron mother corm. (A) Mother corm, (B) initial growth of contractile root at the base of the mother corm (pulling and pushing activity of contractile roots enables mother and daughter corms to move into the ground), and (C) absorbing root of mother corm (about 1
3 mm diameter). From Koocheki, A., Ebrahimian, E., Seyyedi, S.M., 2016. How irrigation rounds and mother corm size control saffron yield, quality, daughter corms behavior and phosphorus uptake. Sci. Hortic. 213, 132
143. FIGURE 7.9 Formation and growth of daughter corms above, below, or at the side of each mother corm. (A) Mother corm, (B) mother corm’s bud (by growing these buds, new daughter corms are formed), (C) formation of new daughter corms below or at the side of the mother corm, (D) formation of contractile root at the base of the new daughter corm, (E) more growth of contractile root, and (F) mother corm’s absorbing roots. From Koocheki, A., Ebrahimian, E., Seyyedi, S.M. 2016a. How irrigation rounds and mother corm size control saffron yield, quality, daughter corms behavior and phosphorus uptake. Sci. Hortic. 213, 132
143.

(Koocheki et al., 2016a; Kumar et al., 2009). Depending on soil microbial, physical and chemical properties, mother corm size and soil moisture content, the depth of penetration of these roots in the soil can be up to 30 cm (Koocheki and Seyyedi, 2015a). The contractile roots (about 3
10 mm diameter), which are usually tuber, fleshy, and white in color pull the plants deeper into the soil with a strong pulling force (Kumar et al., 2009; Rajaei et al., 2009; Zeybek et al., 2012). Being a perennial species (Gresta et al., 2016), the saffron parts that are above and underground grow more from year-to-year (Koocheki et al., 2016b; Yarami and Sepaskhah, 2015). Absorbing and contractile root density (Fig. 7.9) increases from year-to-year with increasing daughter corm formation and density in each year (Nassiri-Mahallati et al., 2007; Sepaskhah and Kamgar-Haghighi, 2009). Yarami et al. (2011) noted that evapotranspiration in saffron increases from year-to-year due to additional corm growth and vegetation. Hence, increases in growth, especially in underground parts, lead to absorbing and contractile root system development (Fig. 7.10) and an increase in the plant’s ability to uptake nutrients in each subsequent year (Seyyedi et al., 2018; Yau et al., 2006). In general, developed aboveground organs stimulate root growth and in turn improve plant capability to uptake water and nutrients (Alizadeh et al., 2009; Asadi et al., 2014; Koocheki et al., 2014). On the other hand, an increase in

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FIGURE 7.10 Saffron compacted root system in the third year. From the third year onward, due to daughter corm increased growth mother and daughter corms appear as a dense and compact mass, making it difficult to distinguish them from each other. Furthermore, root system expansion increases in daughter and mother corms compared with previous years. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

root system expansion improves plant growth and daughter corm formation (Kafi et al., 2002; Renau-Morata et al., 2012). Therefore, the growth of the above- and underground parts of saffron is in line with each other and leads to better absorption of most nutrients from the soil (Gholami et al., 2017; Gresta et al., 2009; Koocheki and Seyyedi, 2016a).

7.2.4 Developmental stages and phonological description Saffron is known as a perennial crop in terms of agronomy but an annual plant in botany (Koocheki and Seyyedi, 2015b). In fact, saffron fields can survive for 6
8 years, depending on agronomic management, soil microbial, physical and chemical properties, or climate parameters (Halvorson, 2008; Helalbeyki et al., 2015; Khademi et al., 2014). Developmental stages and phonological descriptions are based on corm formation and growth (Koocheki and Seyyedi, 2015a). After flowering in November, daughter corms start to form (Koocheki and Seyyedi, 2016b). Later in March, when vegetative growth reaches its maximum, daughter corm formation will be complete (Koocheki and Seyyedi, 2015b; Renau-Morata et al., 2012). All aboveground parts will dry and daughter corms will remain dormant from May to September (Fig. 7.11). During the dormancy period, especially true dormancy, the activity of all antioxidant enzymes in the apical, subapical, and lateral buds of saffron corms, including peroxidase, polyphenol oxidase, superoxide dismutase, and catalase, has been observed to be at the minimum level (Nasirian et al., 2014; Sabet-Teimouri et al., 2010). Hence, saffron fields are free of vegetation during the summer season (Koocheki et al., 2014; Renau-Morata et al., 2012). In general, saffron vegetative growth and stigma yield are typically low in the first year (Feizi et al., 2015; Koocheki and Seyyedi, 2015b). From the second year, increase in saffron yield is achieved, which is due to increasing numbers of daughter corms (Koocheki and Seyyedi, 2015b; Koocheki et al., 2014; Rezvani-Moghaddam et al., 2013b).

7.2.5 Corm nutrient content The saffron corm, the organ intended for planting (Koocheki et al., 2011), contains the storage of nutrient reserves (Kumar et a, 2009; Yau et al., 2006). The nutrient reserves in the mother corms are vital for plant establishment, flowering, and early growth (Koocheki et al., 2014; Mirsafi et al., 2016; Sadeghi et al., 2014). It has been proven that daughter corm growth depends on mother corms until they become independent (Hassanzadeh-Aval et al., 2014; Koocheki and Seyyedi, 2015b). Dynamic nutrients including N and P may be remobilized and stored in the corms, simultaneously with leaves senescence at the end of each year, so they can be reused at the beginning of the next growing season (Gholami et al., 2017; Koocheki et al., 2014). Saffron corm has a set of micro- and macronutrients. N, P, and K contents in saffron corm are recorded as 1.41%, 0.28%, and 0.92%, respectively, on a dry weight basis (Koocheki and Seyyedi; 2015a). The percentage of nutrients in saffron corms is directly related to corm weight (Gresta et al., 2008; Koocheki et al., 2014). An increase in the size of saffron corms results in an increase of nutrients and hence an increase in N and P uptake (Koocheki and Seyyedi, 2019). Nitrogen and phosphorus concentration in large-sized daughter corms increased up to 24% and 36%, respectively, compared with small-sized daughter corms (Koocheki and Seyyedi, 2015b) (Table 7.1).

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FIGURE 7.11 Vegetative stages and phenological description of saffron based on daughter corm growth. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

TABLE 7.1 Effects of mother corm size and year on number of daughter corms and N and P concentration in saffron organs. N concentration (g kg21)

Experimental treatments

Daughter corms 0.1
4 g

4.1
8 g

Over 8g

4 and lower

8.59 a

9.07 d

12.04 d

4.1
8

8.60 a

9.69 c

8.1
12

8.49 a

Over 12

P concentration (g kg21) Aerial part

Daughter corms

Aerial part

0.1
4 g

4.1
8 g

Over 8g

11.59 a

1.64 a

1.85 b

2.41 b

2.01 a

13.24 c

11.47 a

1.68 a

1.93 b

2.51 b

1.96 a

10.32 b

13.99 b

11.61 a

1.67 a

1.98 ab

2.67 ab

2.03 a

8.62 a

10.72 a

14.88 a

11.57 a

1.69 a

2.18 a

2.82 a

1.96 a

First year

8.54 a

9.74 a

12.74 b

11.55 a

1.70 a

1.90 b

2.42 b

1.97 a

Second year

8.62 a

10.16 b

14.34 a

11.57 a

1.64 a

2.07 a

2.78 a

2.01 a

Mother corm size (g)

Year

Values followed by the same letter are not significantly different at P # .05 (DMRT). Source: From Koocheki, A., Seyyedi, S.M., 2015. Relationship between nitrogen and phosphorus use efficiency in saffron (Crocus sativus L.) as affected by mother corm size and fertilization. Ind. Crop. Prod. 71, 128
137.

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FIGURE 7.12 Effects of farm age on nitrogen (A) and phosphorus (B) uptake in saffron daughter corms. Values followed by the same letter are not significantly different at P # .05 (DMRT). From Koocheki, A., Seyyedi, S.M., 2019. Nutrition management and farm’s age affect saffron daughter corms behavior, nutrients uptake and economic water and fertilizer use efficiency: a large scale on-farm experiment in Torbat Heydarieh, Iran. Commun. Soil Sci. Plant Anal. In Press.

Farm age is another factor that can affect the concentration of nutrient reserves in corms (Khademi et al., 2014; Rahimi-Daghi et al., 2015). Koocheki and Seyyedi (2015b) reported that N and P concentration in medium- and largesized daughter corms increased in the second year compared with the first year. In another study (Koocheki and Seyyedi, 2019), N and P percentage in small-, medium-, and large-sized daughter corms increased with increasing farm age from 1 to 4 years old. However, these parameters decreased with increasing farm age from 4 to 6 years (Fig. 7.12). As mentioned before, with the activation of the corm buds, daughter corms can gradually grow (Molina et al., 2004a; Renau-Morata et al., 2012). The growth and differentiation processes in the apical, subapical, and lateral bud meristems usually occurs after flowering, and as a result of cell division, the first daughter corms are formed on the mother corm (Yarami and Sepaskhah, 2015; Yasmin and Nehvi, 2014). This causes a relative increase in the growth of saffron above- and underground organs each year. However, if this phenomenon causes an excessive increase in saffron corms during the perennial lifecycle, it can promote competition over water and nutrients between daughter corms (Gholami et al., 2017; Koocheki and Seyyedi, 2016b; Koocheki et al., 2016a). The imposition of drought stress is another factor that affects the nutrient concentration of saffron corms (Koocheki and Seyyedi, 2016b; Sepaskhah and Kamgar-Haghighi, 2009; Sepaskhah and Yarami, 2009). In general, the nutrient concentration gradually increases due to an increase in the intensity of drought stress (Koocheki and Seyyedi, 2016b). Phosphorus concentration in the saffron parts increases with decreasing irrigation rounds (Koocheki et al.; 2016a) and also reduction in saffron water requirement from 100% to 50% increased phosphorus concentration in daughter corms (Koocheki et al., 2014) (Fig. 7.13). Generally, nutrient and metabolite accumulation along with a reduction in cell volume expansion increase plant resistance against water-deficit stress (Chaves et al., 2002; Ebrahimian and Bybordi, 2011; Wu et al., 2016; Zhang et al., 2017). Drought stress increases protein content in corms, leaves, and roots of saffron (Maleki et al., 2011). Mobile elements, especially phosphorus, can transfer from shoots to underground organs at the end of the growing season before entering into dormancy (Koocheki et al., 2014). Hence, it seems that an increase in nutrient concentration due to limited irrigation is an adaptability mechanism to deal with drought seasons during the perennial lifecycle of saffron.

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FIGURE 7.13 Effects of irrigation regimes on phosphorus concentration in saffron daughter corms. Values followed by the same letter are not significantly different at P # .05 (DMRT). From Koocheki, A., Seyyedi, S.M., Jamshid Eyni, M., 2014. Irrigation levels and dense planting affect flower yield and phosphorus concentration of saffron corms under semi-arid region of Mashhad, Northeast Iran. Sci. Hortic. 180, 147
155.

FIGURE 7.14 Effects of farm age on number of daughter corms (% of total daughter corms). Values followed by the same letter are not significantly different at P # .05 (DMRT). From Koocheki, A., Seyyedi, S.M., 2019. Nutrition management and farm’s age affect saffron daughter corms behavior, nutrients uptake and economic water and fertilizer use efficiency: a large scale on-farm experiment in Torbat Heydarieh, Iran. Commun. Soil Sci. Plant Anal. In Press.

7.2.6 Field age effect on corm production During each growing season, saffron passes its phonological stages by producing daughter corms from each mother corm (Behnia et al., 1999). New corms are generally formed on older corms after flowering (Renau-Morata et al., 2012), so that plant density increases in each growing season (Khademi et al., 2014; Mollafilabi et al., 2015). Accordingly, saffron stigma yield in the first year is usually low and in the fourth to fifth years, the highest stigma yield is recorded. Nonetheless, due to the high number of corms formed in the soil, increased soil stiffness and compaction, and reduced soil fertility, a gradual reduction of flower yield can be observed (Khademi et al., 2014; Koocheki and Seyyedi, 2019; Rahimi-Daghi et al., 2015). Koocheki and Seyyedi (2019) noted that the number of medium- and large-sized daughter corms and weight per m2 increased with increasing farm age from 1 to 4 years. However, these parameters decreased with increasing farm age from 4 to 6 years. The lowest percentage of small-sized daughter corms was also obtained from 4-year-old farms (Fig. 7.14). In a similar study (Mollafilabi et al., 2015), the average number of saffron corms from 1- to 8-year-old farms was recorded as 55, 65, 71, 128, 151, 152, 184, and 248 per m2, respectively, while the highest large-sized corm percentage (over 8 g) was observed from 3-year-old farms (Table 7.2). Increase in saffron flower yield is mainly due to leaf development and the expansion of the root system during the first to fourth growing season (Fig. 7.15). Absorbing and contractile root density increase from year-to-year with increasing daughter corm formation and density in each year (Mollafilabi et al., 2015; Nehvi et al., 2010). Increase in growth, especially underground parts, leads to absorbing and contractile root system development and increase in plants’ ability to uptake nutrients such as phosphorus, which in turn increases phosphorus acquisition efficiency in each consecutive year (Koocheki et al., 2016a).

7.3

Agronomical practices

7.3.1 Corm lifting time and storage As mentioned before, daughter corms become independent of the mother corms after aboveground leaves dry out in May (Koocheki and Seyyedi, 2015a; Nehvi et al., 2010). Each daughter corm continues to grow as a mother corm

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TABLE 7.2 Saffron corm number by farm age. Farm age (year)

Total corm number (m2)

Corm number (%) 8 g and lower

8.1
16 g

16.1
24 g

Over 24 g

1

63.15

29.63

5.43

1.78

55

2

49.41

25.10

14.68

10.8

65

3

40.91

28.55

18.60

11.9

71

4

63.01

27.12

8.10

1.76

128

5

65.91

25.65

6.35

2.07

152

6

47.89

32.97

14.34

4.78

151

7

77.73

19.26

2.76

0.24

184

8

80.11

19.24

0.63

0.00

248

Source: From Mollafilabi, A., Koocheki, A., Rezvani-Moghaddam, P., Nassiri Mahalati, M., 2015. Investigation on the effect of location and field age on yield and frequency of different corm weights of saffron (Crocus sativus L.). Iran. J. Field Crop. Res. 12, 605
612 (in Persian).

FIGURE 7.15 Comparing saffron stand between 1- and 4-yearold farms. In general, saffron vegetative growth is typically low in the first year. From the second year on, an increase in saffron yield is seen, which is primarily due to the increase in the number of daughter corms, additional leaf growth, and expansion of the root system. In the fourth year, saffron produces more roots able to increase the absorption of nutrients, compared to the first year.

during the next growing season (Behnia et al., 1999; Koocheki et al., 2017). Accordingly, mother corms are considered as reproductive organs, that is, seeds (Koocheki et al., 2014). Saffron dormancy starts with a drying phase (Behnia et al., 1999; Kumar et al., 2009). Saffron corms cannot be stored in cold rooms for a long time and prefer to complete their phenological cycles under the soil surface (Koocheki et al., 2016a; Molina et al., 2004b). Hence, poor storage practices may cause serious damage to the corms through increasing respiration and oxidation as well as cause more sensitivity to the activity of pests and diseases (Fig. 7.16), especially fungal pathogens such as Aspergillus niger, Penicillium digitatum, and Rhizopus stolonifera (Saeedizadeh, 2014; Sud et al., 1999). However, irrespective of corm lifting time, the optimal duration of corm incubation before planting was determined to be 30 C for 20 days (Molina et al., 2004b). Reducing storage period from harvesting to planting, controlling storage conditions in terms of temperature, moisture, light, gases, and pests and diseases are considered as the most important measures for saffron corm storage (Molina et al., 2005a; Nassiri-Mahallati et al., 2007). Early true dormancy is the most suitable time for taking daughter corms out of the soil (Koocheki and Seyyedi, 2015a; Koocheki et al., 2016a). Reducing the storage period extends dormancy and allows corms to be able to better establish themselves in new planting beds (Behnia et al., 1999; Molina et al., 2004a). Therefore, a reduction in storage period would increase flowering in the next blooming season (NassiriMahallati et al., 2007; Sadeghi et al., 2003).

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FIGURE 7.16 Saffron corm decay due to poor storage practices. (A) Saffron corm rot (Penicillium digitatum) and (B) corm neck rot (Rhizoctonia crocorum). From Koocheki, A., Seyyedi, S. M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

FIGURE 7.17 The best planting depth for saffron corms. In general, by increasing the mother corm size, planting depth can be slightly increased. From Koocheki, A., Seyyedi, S.M., 2015. Phonological stages and formation of replacement corms of saffron (Crocus sativus L.) during growing period (review article). J. Saffron Res. 3, 134
154 (in Persian).

7.3.2 Planting time Due to reduced storage period, saffron corm planting in the beginning or middle of the true dormancy period is known to induce flower formation (Rostami and Mohammadi, 2013; Sadeghi et al., 2003). During this period, saffron corm growth is suppressed by inhibitor hormones (Koul and Farooq, 1982; Sadeghi et al., 2014), which make farmers able to lift corms out of the soil for future replanting (Ghobadi et al., 2015; Molina et al., 2004b). After this period, the corms start to grow and tissue differentiation occurs. Hence, they are more sensitive to translocation (Koocheki and Seyyedi, 2015a; Sadeghi et al., 2003). Saffron corm planting after terminating dormancy until the end of the summer results in reduced flower yield since flower induction is affected and establishment time reduced (Ghobadi et al., 2015; Koocheki and Seyyedi, 2015b; Rostami and Mohammadi, 2013). Therefore, the best time for daughter corm planting is sometime between daughter corm harvesting and the middle of the true dormancy period (early July).

7.3.3 Planting depth Corm planting depth plays a key role in flowering and daughter corm growth. Planting depth depends on mother corm weight; a depth of 12
15 cm is recommended for 8.1
12 g corms (Koocheki et al., 2011). By increasing or decreasing the mother corm size, planting depth can be slightly changed (Fig. 7.17).

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FIGURE 7.18 Saffron corms planting using basin method at 100 corms m22 and 25 cm distance between planting rows. From Koocheki, A., Rezvani-Moghaddam, P., Seyyedi, S.M., 2019. Saffron-pumpkin/ watermelon: a clean and sustainable strategy for increasing economic land equivalent ratio under limited irrigation. J. Clean. Prod. 208, 1327
1338.

Deeper planting causes a significant reduction in flowering, especially for buds located on the base of the corms (De Juan et al., 2009; Gerdakaneh et al., 2017). Most of the daughter corms are generated from the subapical or lateral buds of the mother corms, so it is not surprising that an increase in planting depth is a reason for a reduced number of daughter corms (Aghazadeh and Hemmatzadeh, 2012; Koocheki and Seyyedi, 2015a). On the other hand, surface planting can cause serious injury to corms due to heat stress in summer or cold stress in winter (De Juan et al., 2009; Gerdakaneh et al., 2017).

7.3.4 Row spacing, corm density In addition to planting depth, the planting pattern and mother corm density are considered as crucial factors determining daughter corm generation and flower yield (De Juan et al., 2009; Koocheki et al., 2011; Nazarian et al., 2016). In the row planting method, 75
100 corms m22 with 25 cm distance is recommended (Fig. 7.18). Depending on mother corm size, 8
13 tons ha21 of corm is needed to reach to this planting density (Koocheki et al., 2011). Even when using the recommended planting pattern and density, saffron yield in the first year of cultivation is usually low, but from the second year, it increases due to daughter corm formation (Khademi et al., 2014; Koocheki et al., 2016a; Mollafilabi et al., 2015). Therefore, saffron cultivation at low densities (conventional practice) is not economically feasible in the early years (Koocheki et al., 2011; Rezvani-Moghaddam et al., 2013b). Hence, higher planting densities, called dense corm planting, are considered as an alternative approach and planting pattern to offset yield loss during early years (Koocheki et al., 2014). Higher planting densities help saffron to use growth resources such as nutrients efficiently. Dense planting up to 400 corms m22 (Fig. 7.19) was investigated by Koocheki et al. (2012). In another study, Koocheki et al. (2011) investigated different planting densities and found that an increase in planting density (from 4 to 12 tons corms per hectare) leads to an increase in flower number and stigma yield.

7.3.5 Corm size/weight Saffron corm is known as a source of food reserves (Koocheki and Seyyedi, 2015a; Maggio et al., 2006). With drying aboveground parts, the beginning of the dormancy period, the nutrients are remobilized to the underground organs (Akrami et al., 2014; Koocheki et al., 2014). Hence, nutrient reserves in mother corms determine saffron growth, especially during early growth stages (Hassanzadeh-Aval et al., 2014; Sahabi et al., 2017). In general, heavier mother corms supply more energy to daughter corms (Douglas et al., 2014; Koocheki et al., 2014). Thus, mother corms with appropriate weight can improve plant regrowth and yield (Alizadeh-Salteh, 2016; De Juan et al., 2009; Renau-Morata et al., 2012). It has been proven that daughter corm growth depends on the mother corm until they become independent; consequently, mother corm size has a significant impact on daughter corm formation (Koocheki et al., 2014; Renau-Morata et al., 2012). Accordingly, the greater amount of food reserves in mother corms can increase flower yield by increasing plant growth and fibrous and contractile root expansion (Amirian and Kargar, 2016; Koocheki et al., 2016a; Tavakkoli-Kakhki et al., 2016). Large-sized mother corms produce plants with more leaf area than those grown from small corms as a result of more carbohydrate reserves (Alizadeh-Salteh, 2016; Hassanzadeh-Aval et al., 2014). These plants are able to uptake

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FIGURE 7.19 Interaction effects of corm density and manure application on flower number (A) and dried stigma yield (B) in saffron. Values followed by the same letter are not significantly different at P # .05 (DMRT). From Koocheki, A., RezvaniMoghaddam, P., Mollafilabi, A., Seyyedi, S. M., 2012. Effects of high corm planting density and applying manure on flower and corm yields of saffron (Crocus sativus L.). Fourth International Saffron Symposium: Advanced in Saffron Biology Technology and Trade. 22
25 October 2012, Kashmir.

TABLE 7.3 Effects of mother corm size on number of saffron daughter corms. Number of daughter corms (m2)

Experimental treatments 0.1
4 g

4.1
8 g

Over 8 g

Total

99.94 (81.0) d

17.89 (15.1) d

4.89 (7.3) d

122.72 d

Mother corm size (g) 4 and lower 4.1
8

111.28 (71.1) c

34.89 (22.7) c

10.17 (6.9) c

156.33 c

8.1
12

120.72 (66.2) b

50.00 (26.9) b

13.00 (6.2) b

183.72 b

Over 12

145.83 (63.7) a

66.89 (29.0) a

17.72 (3.9) a

230.44 a

Values followed by the same letter are not significantly different at P # .05 (DMRT). The number in parenthesis indicated the percentage of daughter corms from total daughter corms. Source: From Koocheki, A., Seyyedi, S.M., 2015. Relationship between nitrogen and phosphorus use efficiency in saffron (Crocus sativus L.) as affected by mother corm size and fertilization. Ind. Crop. Prod. 71, 128
137.

minerals from the soil and produce more daughter corms at the end of the growing season (Douglas et al., 2014; Koocheki and Seyyedi, 2016a). In fact, smaller corms produce plants with low growth rate and smaller leaf area compared with larger corms (Gresta et al., 2008; Khavari et al., 2016; Nassiri-Mahallati et al., 2007). Increase in leaf area, root dry weight, and active bud number in saffron corm due to increased mother corm size from 4 to 12 g has been confirmed by Koocheki et al. (2007). Similarly, the medium- (4.1
8 g) and large-sized (over 8 g) daughter corms number increased with increasing mother corm size (Table 7.3). Koocheki et al. (2016a) noted that the maximum flower number and stigma yield were obtained when large-sized ( . 10 g) mother corms were planted (Fig. 7.20A,B). The direct relationship between mother corm size and saffron flower yield has been reported by numerous researchers (De Mastro and Ruta, 1993; Gresta et al., 2008; HassanzadehAval et al., 2014; Kumar et al., 2009). In one study (Table 7.4), N acquisition efficiency, N use efficiency, N harvest index, P acquisition efficiency, P use efficiency, and P harvest index significantly improved by increasing mother corm size (Koocheki and Seyyedi, 2015b). Generally, large-sized daughter corms are more important than small ones because small-sized daughter corms (4 g and lower) lead to less or no flower production (Gresta et al., 2008; Koocheki et al., 2014; Kumar et al., 2009). Considering the positive role of N and P in improving flower and corm yields (Chaji et al., 2013; Omidi et al., 2009),

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FIGURE 7.20 Interaction effects of year and mother corm size on flower number (A) and dried stigma yield (B) in saffron. Values followed by the same letter are not significantly different at P # .05 (DMRT). From Koocheki, A., Ebrahimian, E., Seyyedi, S.M., 2016. How irrigation rounds and mother corm size control saffron yield, quality, daughter corms behavior and phosphorus uptake. Sci. Hortic. 213, 132
143.

TABLE 7.4 Interaction effects of year and mother corm size on nitrogen and phosphorus uptake in saffron. Year

Mother corm size (g)

N acquisition efficiency (%)

N use efficiency (g g21)

N harvest index (%)

P acquisition efficiency (%)

P use efficiency (g g21)

P harvest index (%)

First year

4 and lower

16.87 g

6.29 f

38.35 e

8.76 f

3.49 f

40.96 de

4.1
8

20.92 f

10.51 e

50.42 cd

11.13 e

6.03 e

55.03 bc

8.1
12

24.88 e

13.59 d

54.16 bc

13.60 d

7.75 d

56.60 bc

Over 12

30.26 d

18.30 c

60.93 a

16.94 c

10.88 c

64.82 a

4 and lower

28.19 d

9.19 e

32.72 f

14.29 d

5.17 e

36.16 e

4.1
8

34.37 c

14.43 d

40.89 e

18.36 c

8.10 d

43.34 d

8.1
12

42.93 b

21.70 b

49.62 d

23.66 b

12.60 b

52.23 c

Over 12

50.85 a

28.90 a

55.92 b

28.98 a

17.55 a

59.37 ab

Second year

Values followed by the same letter are not significantly different at P # .05 (DMRT). The number in parentheses indicates the percentage of phosphorus content from the total plant. From Koocheki, A., Seyyedi, S.M., 2015. Relationship between nitrogen and phosphorus use efficiency in saffron (Crocus sativus L.) as affected by mother corm size and fertilization. Ind. Crop. Prod. 71, 128
137.

an increase in the concentration of these elements, especially in large-sized daughter corms (over 8 g), could be an effective way to increase saffron yield. A positive correlation between N and P concentration in daughter corms (Fig. 7.21A) and between P and N harvest index (Fig. 7.21B) represents the importance of balanced nutrition in daughter corm growth and saffron production.

7.3.6 Corm size classification Despite the importance of corm size in the flowering process of saffron, there is no uniform and precise index for corm classification. Moreover, the criteria presented in the available literature are somewhat different. For example,

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107

FIGURE 7.21 Relationship between (A) nitrogen (N) concentration and phosphorus (P) concentration in daughter corms and (B) N harvest index and phosphorus (P) harvest index in saffron. **Statistical differences at P # 0.01. From Koocheki, A., Seyyedi, S.M., 2015. Relationship between nitrogen and phosphorus use efficiency in saffron (Crocus sativus L.) as affected by mother corm size and fertilization. Ind. Crop. Prod. 71, 128
137.

FIGURE 7.22 Saffron corm sorting based on the weight value criterion. From Koocheki, A., Seyyedi, S.M., 2015. Relationship between nitrogen and phosphorus use efficiency in saffron (Crocus sativus L.) as affected by mother corm size and fertilization. Ind. Crop. Prod. 71, 128
137.

Ghobadi et al. (2015) considered 5
9 g mother corms as small-sized and 10
14 g mother corms as large-sized. Mollafilabi et al. (2015) divided saffron corms into four groups: small (8 g and lower), medium (1.8
16 g), large (16.1
24 g), and very large (over 24 g). In another study, Nassiri-Mahallati et al. (2007) identified 3 and 7 g mother corms as small-sized and large-sized, respectively. In order to provide clear and precise criteria for classification of produced saffron corms, corm sorting has been proposed based on weight rather than diameter (De Mastro and Ruta, 1993), as it is more accurate and practical (Koocheki et al., 2016a). The weighing value is considered based on corm moisture content (16% plus corm tunics). As can be seen from Fig. 7.22 this sorting is as follows (Koocheki and Seyyedi, 2015b): (1) small-sized (4 g and lower), (2) medium-sized (4.1
8 g), (3) relatively large-sized (1.8
12 g), and (4) large-sized (over 12 g). It is possible to produce corms over 12 g under field conditions (Mollafilabi et al., 2015; Seyyedi et al., 2018). Nonetheless, given that saffron is basically cultivated in arid and semiarid regions (Sepaskhah and Kamgar-Haghighi, 2009), over 12 g mother corms contain a very small proportion of the total produced corms (Koocheki and Seyyedi, 2016b; Rezvani-Moghaddam et al., 2013b). Hence, corms weight more than 12 g are considered as large-sized (Koocheki and Seyyedi, 2015b). Generally, there is a direct relationship between corm diameter and corm weight, so that corms weighing about 10 g are 30 mm in diameter (Mollafilabi et al., 2015). Nonetheless, as stated, the criteria for the classification of mother corms based on diameter value is less important as it is not very practical.

7.3.7 Planting beds 7.3.7.1 Application of organic and chemical fertilizers In general, heavy soils with high clay and low sand content can reduce the growth of daughter corms. However the loose, low density, and well drained soils with high organic content are considered suitable for saffron production (Kumar et al., 2009; Lage and Cantrell, 2009). Aghhavani-Shajari et al. (2015) reported that light soil texture has more advantages than heavy soil texture in saffron cultivation. Therefore, soil amendments can improve saffron flower and corm yields (Akrami et al., 2014; Madahi et al., 2017; Mollafilabi and Khorramdel, 2016; Rezvani-Moghaddam et al., 2013a; Teimori et al., 2013).

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TABLE 7.5 Effects of type of fertilizer on number of daughter corms and N and P concentration of daughter corms. Type of fertilizer

Number of daughter corms (m2)

N concentration in daughter corms (g kg21)

P concentration in daughter corms (g kg21)

0.1
4 g

4.1
8 g

Over 8g

0.1
4 g

4.1
8 g

Over 8g

0.1
4 g

4.1
8 g

Over 8g

Manure

107.00 c

54.25 a

17.58 a

8.65 a

10.63 a

15.42 a

1.67 a

2.27 a

2.92 a

Chemical

118.75 b

44.25 b

10.58 b

8.51 a

10.01 b

13.49 b

1.72 a

1.96 b

2.58 b

Control

132.58 a

28.75 c

6.17 c

8.58 a

9.21 c

11.70 c

1.63 a

1.72 c

2.30 c

Values followed by the same letter are not significantly different at P # .05 (DMRT). Source: From Koocheki, A., Seyyedi, S.M., 2015. Relationship between nitrogen and phosphorus use efficiency in saffron (Crocus sativus L.) as affected by mother corm size and fertilization. Ind. Crop. Prod. 71, 128
137.

The lifecycle of saffron, as a perennial plant under field conditions, can change the physical and chemical properties of soil in the long term (Helalbeyki et al., 2015; Khademi et al., 2014; Maleki et al., 2017). Unlike chemical fertilizers, which have an adverse effect on soil biology and structure (Savci, 2012), organic fertilizers improve the physical, chemical, and biological properties of the soil (Celik et al., 2004; Gracey, 1984; Qiu et al., 2016). The positive role of manure in increasing flower and stigma yield was reported by Amiri (2008) who stated that manure application improves the physical and chemical properties of soil and increases the cation exchange capacity and N, K, and Ca uptake by saffron plants. According to Koocheki and Seyyedi (2015b), large-sized daughter corms number and N and P concentration of daughter corms in composted cattle manure were significantly higher than chemical fertilizer (Table 7.5). Behdani et al. (2017) noted that an organic production system had a significant positive effect on the growth of saffron daughter corms and number of flowering buds when compared to conventional production systems. Similar results have been reported by other researchers (Alipoor-Miandehi et al., 2014; Rezvani-Moghaddam et al., 2013a; Yarami and Sepaskhah, 2015). Application of organic fertilizers in saffron production is essentially dependent on the amount of organic matter in the soil (Behdani et al., 2006; Mollafilabi and Khorramdel, 2016; Rezvani-Moghaddam et al., 2013a). In general, the use of organic fertilizers in saffron farms is important for these reasons: 1. As noted, the distribution of saffron farms is mainly in arid and semiarid regions. However, soil organic matter deficiency is one of the most important factors limiting cultivation in these areas (Behdani et al., 2006; Seyyedi et al., 2015). Annual rainfall shortage, poor vegetation, and frequent harvesting are among the most important reasons for reducing soil organic matter content (Koocheki and Seyyedi, 2015b; Rezvani-Moghaddam et al., 2015). 2. The perennial lifecycle of saffron under the field conditions is another factor that leads to a decrease in the amount of soil organic matter, especially in the third year onward (Koocheki et al., 2017, 2018; Rabani-Foroutagheh et al., 2013). In fact, multiyear production in saffron farms is the result of plant cultivation in the first year. Therefore, application of organic fertilizer after plant establishment is practically difficult (Ahmadi et al., 2017). 3. Changes in soil compaction in response to agricultural operations, especially after the third growing season, is a common phenomenon in saffron farms (Koocheki et al., 2017; Rahimi-Daghi et al., 2015). In general, for each unit of reduction in soil organic matter content, the negative effects of soil compaction on the growth of the corms are equally increased (Koocheki and Seyyedi, 2015b; Madahi et al., 2017; Mollafilabi and Khorramdel, 2016).

7.3.7.2 Crop residue Based on phonological stages, there is no vegetation in saffron fields during the summer season (Mollafilabi et al., 2015). On the other hand, the optimum temperature for flower induction in saffron is between 23 C and 27 C (Molina et al., 2005b). Hence, nutrients loss due to soil erosion, increased soil temperature, and reduced flower initiation are the most obvious impacts and problems of saffron cultivation occurring during the saffron dormancy period (Koocheki et al., 2019; Rezvani-Moghaddam et al., 2013b).

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FIGURE 7.23 Saffron flowering (in October) after applying the wheat straw mulch (in June). Generally, retaining wheat residue improves the physical characteristics of soil and facilitates flower emergence. Source: From Rezvani-Moghaddam, P., Koocheki, A., Mollafilabi, A., Seyyedi, M., 2013. The effects of different levels of applied wheat straw in different dates on saffron (Crocus sativus L.) daughter corms and flower initiation criteria in the second year. Saffron Agron. Technol. 1, 55
70 (in Persian).

In order to increase the production and stability of saffron cropping systems, green cover or companion crop residue can be used to effectively reduce the adverse effects of high temperatures on dormant daughter corms (Koocheki et al., 2019; Shabahang et al., 2013). Furthermore, retaining crop residue improves soil physical characteristics and facilitates flower emergence (Aghhavani-Shajari et al., 2017; Rezvani-Moghaddam et al., 2013b; Shabahang et al., 2013). During the process of residue decomposition, considerable amounts of mineral nutrients are released into the soil (BlancoCanqui and Lal, 2009; Butterly et al., 2011; De Gryze et al., 2005), which can be recycled by the saffron roots (Koocheki et al., 2017). The use of wheat residue is one of the most effective and recommended methods in saffron cultivation. It can be spread on the soil surface at the beginning of the saffron dormancy period (Fig. 7.23). Retaining wheat residue during the saffron dormancy period can accelerate the flowering process and increase the number of large-sized daughter corms (Table 7.6) (Rezvani-Moghaddam et al., 2013b). The amount of N and P in the wheat residue are 0.47% and 0.27%, respectively (Ebrahimian et al., 2016), which can be recycled during the perennial lifecycle of saffron (Koocheki and Seyyedi, 2019; Madahi et al., 2017). Moreover, reducing soil erosion, increasing organic matter content, and improving soil permeability are among the other benefits of wheat mulch application (Bastian et al., 2009; Bruce et al., 2005; Du Preez et al., 2001).

7.3.7.3 Intercropping In addition to plant mulches, proper implementation of intercropping, especially with medicinal and aromatic plants, helps to cover soil and avoid loss of water and nutrients (Rezvani-Moghaddam et al., 2014; Singh et al., 2010; Sujatha et al., 2011). In order to successfully develop the intercropping systems in saffron cultivation, the candidate species should essentially have the same ecophysiological requirements as saffron (Asadi et al., 2016; Khorramdel et al., 2016). For instance, cumin (Cuminum cyminum L.) is an annual and herbaceous plant from the Apiaceae family (Bettaieb Rebey et al., 2012) that is cultivated in arid and semiarid regions of Iran as a medicinal plant (Alinian and Razmjoo, 2014). Cumin plants grow to 22
30 cm tall and are drought-tolerant (Alinian and Razmjoo, 2014; Alinian et al., 2016) so can be intercropped with saffron. Furthermore, saffron intercropping with other crops, including Cucurbitaceae and Poaceae families, may economically and environmentally be feasible and can be considered to neutralize some environmental adverse impacts (Koocheki et al., 2016b, 2009; Naderidarbaghshahi et al., 2013). Watermelon and pumpkin are the two most commonly cultivated crops in semiarid regions (Choopan et al., 2014; Erdem and Yuksel, 2003). They are suitable to be intercropped with saffron as their lifecycle does not overlap (Koocheki et al., 2019). Furthermore, watermelon and pumpkin make excellent groundcover (Fandika et al., 2011; Olasantan, 2007) and act as a live mulch to hold moisture in the soil and keep the soil cool during warm seasons (Koocheki et al., 2019; Soltani et al., 1995). Consequently, saffronwatermelon or saffron-pumpkin intercropping (Fig. 7.24) can provide considerable profitability in both spatial and temporal dimensions (Koocheki et al., 2019).

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TABLE 7.6 Interaction effects of applying dates and wheat mulch levels on some characteristics of saffron daughter corms. Application dates

June

August

October

Wheat mulch levels (t ha21)

Number of daughter corm (m22) 0.1
4 g

4.1
8 g

8.1
12 g

More than 12 g

0

77.3 cd

17.0 f-h

7.3 f

6.7 def

2

55.0 def

16.6 fgh

8.7 ef

7.7 cde

4

94.3 bc

34.0 b

8.3 f

5.3 efg

6

106.7 b

30.3 bcd

16.7 c

10.3 abc

8

91.7 bc

31.3 bc

18.0 bc

12.0 a

0

59.0 def

10.6 h

9.3 def

7.7 cde

2

36.7 f

11.3 h

12.0 de

5.0 efg

4

75.0 cde

24.7 c-f

9.3 def

4.7 fg

6

97.6 bc

23.3 c-f

20.3 b

11.3 ab

8

74.6 cde

27.0 bcd

16.7 c

9.0 bcd

0

54.6 def

17.7 f-h

9.3 f

9.0 bcd

2

148.3 a

14.3 gh

10.7 def

3.0 g

4

55.0 def

25.3 b-f

7.3 f

4.7 fg

6

98.0 bc

22.0 d-g

12.3 d

11.0 ab

8

109.7 b

45.0 a

28.0 a

11.0 ab

Values followed by the same letter are not significantly different at P # .05 (DMRT). Source: From Rezvani-Moghaddam, P., Koocheki, A., Mollafilabi, A., Seyyedi, M., 2013b. The effects of different levels of applied wheat straw in different dates on saffron (Crocus sativus L.) daughter corms and flower initiation criteria in the second year. Saffron Agron. Technol. 1, 55
70 (in Persian).

FIGURE 7.24 Saffron flowering onto pumpkin (A) and watermelon (B) aboveground parts in autumn after passing dormancy period. From Koocheki, A., Rezvani-Moghaddam, P., Seyyedi, S. M., 2019. Saffron-pumpkin/watermelon: a clean and sustainable strategy for increasing economic land equivalent ratio under limited irrigation. J. Clean. Prod. 208, 1327
1338.

The implementation of intercropping systems based on saffron-ajwain (Koocheki et al., 2009), saffron-chamomile (Naderidarbaghshahi et al., 2013), and saffron-black seed (Koocheki et al., 2009) has also shown positive results. Moreover, the successful cultivation of saffron between apple trees based on the agroforestry system has also been considered (Moosavi et al., 2014).

7.3.7.4 Planting under controlled environment Where saffron cultivation is faced with barriers and restrictions under field conditions, production under controlled conditions is considered as an alternative approach (Koocheki and Seyyedi, 2016a; Sabet-Teimouri et al., 2010).

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FIGURE 7.25 Saffron corm planting under a controlled environment. (A) Saffron corm planting into the perlite, vermiculite, and coco peat substrates and (B) saffron production based on soilless plant culture using hydroponics system. From Mollafilabi, A., Koocheki, A., Rezvani-Moghaddam, P., Nassiri-Mahallati, M., 2014. Effect of plant density and corm weight on yield and yield components of saffron (Crocus sativus L.) under soil, hydroponic and plastic tunnel cultivation. Saffron Agron. Technol. 1, 14
28 (in Persian).

The production of saffron in controlled environments, especially in modern greenhouses, is possible by preparing optimum culture media using commercial substrates (Mollafilabi et al., 2014, 2013). These substrates include solid compounds that are free of pathogens and can supply micro- and macronutrients during the growing season (Packer and Clay, 2000; Yasmin and Nehvi, 2014). On the other hand, the commercial substrates have the physically stable structure (Fuller et al., 2009; Yang et al., 2017) and, from a chemical point of view, are composed of relatively neutral compounds (Malandrino et al., 2006; Souret and Weathers, 2000). Among the organic and inorganic substrates used for saffron production are peat moss, perlite, vermiculite, and coco peat (Fig. 7.25A). In addition to commercial substrates, the production of saffron based on soilless plant culture using hydroponic or aeroponic systems (Fig. 7.25B), as alternative approaches, can be implemented (Mollafilabi et al., 2014, 2013). In general, the accurate adjustment of temperature, humidity, and light (Souret and Weathers, 2000; Yasmin and Nehvi, 2014), saffron corm planting in high densities (Maggio et al., 2006), the production of flowers in shorter periods of time compared with production under field conditions (Mollafilabi et al., 2013; Sorooshzadeh and Tabibzadeh, 2015), the feasibility of controlling pests and pathogens in a simpler and faster pattern (Maggio et al., 2006;

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Renau-Morata et al., 2013), as well as the availability of conditions for accurate management of water and nutrients (Sorooshzadeh and Tabibzadeh, 2015; Yasmin et al., 2013) are considered as positive aspects of soilless saffron culture. Souret and Weathers (2000) noted that saffron corms grown aeroponically and hydroponically produced more flowers and leaves compared with corms from soil-grown control plants.

7.3.8 Application of hormones Plant hormones (including auxin, gibberellin, cytokinin, and ethylene) are produced in very small amounts in certain parts of plants and transferred to other parts (Campos-Rivero et al., 2017; Zhang et al., 2009). The role of plant hormones in the saffron plant involves biochemical, physiological, and morphological responses during the perennial lifecycle (Amirshekari et al., 2007; Aytekin and Acikgoz, 2008). The development of the root system, the induction of flowering, and the stimulation of growth in the above- and underground parts have been identified as some of the positive effects of the external application of hormones in saffron production (Amirian and Kargar, 2016; Azizbekova et al., 1978; Koul and Farooq, 1982). For instance, gibberellic acid can reduce the potential of water in the cell and cause more water to enter the cell and increase its volume by condensing the cell sap through starch-to-sugar hydrolysis (Konishi et al., 2005; Ohkawa et al., 1989; Xu et al., 2016). Therefore, due to these effects, it is possible to increase root dry weight (Amirshekari et al., 2007; Komatsu and Konishi, 2005). Application of gibberellin on dormant corms has resulted in a decrease in sprouting buds and hence fewer daughter corms. In this case, apical buds produced larger daughter corms (Amirshekari et al., 2007; Greenberg-Kaslasi, 1991). Moreover, the positive role of gibberellin on flowering induction has also been reported (Amirian and Kargar, 2016).

7.4

Corm and climate change

Establishment and differentiation of reproductive organs to the buds (late August to early September), organogenesis (late September), and reproductive organ growth into the buds (late September to early October) are some of the saffron development stages prior to flowering (Kafi et al., 2002). The optimum temperature for flower induction in saffron is 23 C
27 C (Molina et al., 2005b). The appropriate temperature for flowering has also been reported in the range of 15 C
17 C (Molina et al., 2005b). Hence, flower induction (early summer) until flowering is directly affected by temperature (Arsalani et al., 2015; Koocheki et al., 2010; Molina et al., 2005a) so that high temperatures during summer negatively affect flowering in saffron (Ensaf et al., 2015; Koocheki et al., 2019). The consequences of climate change have largely focused on global warming (Ming et al., 2014; Wernberg et al., 2011). Global warming, due to increased anthropogenic greenhouse gas emissions (Guardia et al., 2016; Zhao, 2011), may also have a negative impact on the flowering process of saffron (Jafarzadeh et al., 2015; Koocheki et al., 2010). As mentioned earlier, the physiological and biochemical mechanisms associated with bud stimulation (during flower induction until flower appearance) are basically influenced by temperature (Abrishamchi, 2003). Hence, an increase in the relative environmental temperature could possibly disrupt the process of differentiation in vegetative-reproductive sprouts (Arsalani et al., 2015; Fernandez, 2004; Molina et al., 2005b). Saffron flower emergence may be delayed due to an increase in average air temperature during autumn (Koocheki and Seyyedi, 2015a). Due to an increase in the average air temperature (between 1.5 C and 2 C, Table 7.7), the date of saffron flower appearance (about mid-October) TABLE 7.7 Increasing the air temperature and possible dates for flowering in saffron based on climatic conditions in Khorasan-Razavi province, Iran. An increase in average air temperature ( C)

Possible dates for flowering in saffron Minimum date

Maximum date

0.5

20th of October

1st of November

1.0

3rd of November

14th of November

1.5

16th of November

28th of November

2.0

30th of November

13th of December

Source: From Koocheki, A., Nassiri, M., Alizadeh, A., Ganjali, A., 2010. Modelling the impact of climate change on flowering behavior of Saffron (Crocus sativus L.). Iran. J. Field Crop. Res. 7, 583
594 (in Persian).

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in Khorasan-Razavi Province, Northeast Iran is postponed until late December (Koocheki et al., 2010). This postponement can also occur in other areas with similar climates. Furthermore, saffron is known as a hysteranthous plant, so that under optimal temperature conditions, its flowers start to appear before the leaves emerge (Koocheki et al., 2016a). Nevertheless, a relative increase in air temperature causes the saffron flowers to appear after the leaves emerge (Koocheki and Seyyedi, 2015a; Molina et al., 2005b). As a result, this phenological undesirable process can disrupt the flower picking through the growth of leaves at inappropriate times (Jafarzadeh et al., 2015; Koocheki et al., 2019). The length of the vegetative growth period will be shortened by any delay at the beginning of the reproductive growth period (Koocheki and Seyyedi, 2015a). Hence, reduced saffron corm weight can be considered as another consequence of climate change. Fluctuations in precipitation patterns (Djebou and Singh, 2016), reducing water resource quality (Bazrafshan et al., 2019; Fan and Shibata, 2015), and drought-induced water scarcity (Kahil et al., 2015), other negative climatic and environmental outcomes, can reduce saffron flower and corm yields. The adoption of strategies to reduce the negative effects of climate change and adaptation to climatic conditions in the areas of saffron cultivation could be effective in reducing these adverse effects (Jafarzadeh et al., 2015; Koocheki et al., 2010).

7.5

Conclusion

Saffron, a perennial and autumn-flowering geophyte plant under field conditions, is a crop suitable for arid and semiarid areas that survives for 6
8 consecutive years, depending on agronomic management or climate parameters. A variety of factors can affect the multiyear production cycle of saffron. In addition to optimal temperature conditions for differentiation of reproductive organ and flower emergence, saffron yield is fundamentally determined based on correct agronomic management and includes: (1) selecting the appropriate mother corms; (2) selecting healthy saffron corms without symptoms caused by physical damages, pests, and pathogens; (3) reducing storage period from harvesting to replanting; (4) properly storing corms in terms of controlling temperature, humidity, and pest and pathogen activity; (5) planting corms at the beginning of the true dormancy period; (6) paying attention to the benefits of dense corm planting; (7) preparing soil based on organic fertilizer application; (8) retaining green cover or plant residue on the soil surface during summer; (9) produced saffron based on intercropping patterns; and (10) using modern aspects of saffron production based on soilless plant culture. The above can play an indelible and irrefutable role in increasing the production and profitability of saffron. It is also important to change the view of saffron producers. While dried stigma is sold as the main product of saffron in commercial markets, all agronomic practices under field conditions should focus on the production of standard corms. Consequently, the production of standard corms should be recognized as the final product of the saffron perennial lifecycle.

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27 (in Persian). Nehvi, F.A., Lone, A.A., Khan, M.A., Maqhdoomi, M.I., 2010. Comparative study on effect of nutrient management on growth and yield of saffron under temperate conditions of Kashmir. Acta Hortic. 850, 165
170. Ohkawa, H., Kamada, H., Sudo, H., Harada, H., 1989. Effects of gibberellic acid on hairy root growth in Datura innoxia. J. Plant Physiol. 134, 633
636. Olasantan, F.O., 2007. Effect of population density and sowing date of pumpkin on soil hydrothermal regime, weed control and crop growth in a yam
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380. Omidi, H., Naghdibadi, H.A., Golzad, A., Torabi, H., Fotoukian, M.H., 2009. The effect of chemical and bio-fertilizer source of nitrogen on qualitative and quantitative yield of saffron (Crocus sativus L.). J. Med. Plant 8, 98
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17 (in Persian). Rajaei, S.M., Niknam, V., Seyyedi, S.M., Ebrahimzadeh, H., Razavi, K., 2009. Contractile roots are the most sensitive organ in Crocus sativus to salt stress. Biol. Plant. 53, 523
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46. Renau-Morata, B., Moya´, L., Nebauer, S.G., Seguı´-Simarro, J.M., Parra-Vega, V., Go´mez, M.D., et al., 2013. The use of corms produced under storage at low temperatures as a source of explants for the in vitro propagation of saffron reduces contamination levels and increases multiplication rates. Ind. Crop. Prod. 46, 97
104. Rezvani-Moghaddam, P., Khorramdel, S., Amin Ghafori, A., Shabahang, J., 2013a. Evaluation of growth and yield of saffron (Crocus sativus L.) affected by spent mushroom compost and corm density. J. Saffron Res. 1, 13
26 (in Persian). Rezvani-Moghaddam, P., Koocheki, A., Mollafilabi, A., Seyyedi, M., 2013b. The effects of different levels of applied wheat straw in different dates on saffron (Crocus sativus L.) daughter corms and flower initiation criteria in the second year. Saffron Agron. Technol. 1, 55
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203 (in Persian). Rostami, M., Mohammadi, H., 2013. Effects of planting date and corm density on growth and yield of saffron (Crocus sativus L.) under Malayer climatic conditions. J. Agroecol. 5, 27
38 (in Persian). ´ ., Go´mez-Go´mez, L., Trapero, A., Castro-Dı´az, N., Ahrazem, O., 2013. Saffron corm as a natural source of fungicides: the role of Rubio-Moraga, A saponins in the underground. Ind. Crop. Prod. 49, 915
921. Sabet-Teimouri, M., Kafi, M., Avarseji, Z., Orooji, K., 2010. Effect of drought stress, corm size and corm tunic on morphoecophysiological characteristics of saffron (Crocus sativus L.) in greenhouse conditions. J. Agroecol. 2, 323
334 (in Persian). Sadeghi, B., Aghamiri, A., Negari, K., 2003. Effect of summer irrigation on saffron flowering. In: Hemmati-Kakhki, A., Allahyari, M. (Eds.), Proceedings of the Third National Symposium on Saffron, 2
3 December 2003, Mashhad, Iran (in Persian). Sadeghi, S.M., Dehnadi-Moghaddam, G., Dooroodian, H., 2014. Evaluation of effects of date, depth and corm sowing distance on corms growth and stigma yield of saffron (Crocus sativus L.) in Langarood. Guilan Province. Saffron Agron. Technol. 2, 137
144 (in Persian).

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Saeedizadeh, A., 2014. Identification of some saffron corm rot fungi and their control. Saffron Agron. Technol. 2, 205
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1992.

Chapter 8

Ecophysiology of saffron Parviz Rezvani-Moghaddam Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 8.1 Introduction 8.2 Climatic factors for crop production 8.2.1 Temperature 8.2.2 Precipitation 8.3 Lifecycle 8.3.1 Flowering phase 8.3.2 Vegetative phase 8.3.3 Production of replacement corms 8.3.4 Dormant phase 8.4 Growth parameters 8.4.1 Leaf area index 8.4.2 Crop growth rate

8.1

119 120 120 121 122 122 122 122 123 123 125 126

8.4.3 Relative growth rate 8.4.4 Net assimilate rate 8.4.5 Leaf area ratio 8.4.6 Leaf weight ratio 8.4.7 Corms 8.4.8 Whole plant 8.4.9 Source and sink relationship in the growth organs 8.5 Effects of environmental changes on the quality of saffron 8.6 Yield determination 8.7 Conclusion References

126 130 131 131 133 133 134 134 135 135 135

Introduction

Saffron (Crocus sativus) is known for its unique ecological and agroecological characteristics as an important plant in water-restricted areas (Azizi-Zohan et al., 2008). Due to the ecological characteristics of the saffron niches, which is comparable to other crops (Koocheki, 2004), it is resistant to drought (Azizi-Zohan et al., 2008) and hence less water is required for its production. Therefore it plays a significant role in the economic and social aspects of arid and semiarid regions such as Iran, particularly in the Khorasan Razavi and Southern Khorasan provinces (Behdani and Fallahi, 2015). The growth of saffron is a unique process, in which the flowers appear in the autumn before the onset of growth, and afterward, the leaves appear and expand. In the next stage the new corms form and initiate and evolve the flower (Kafi, 2006). Therefore the most suitable selection of corms for cultivation, identification of the date of sowing and the optimum crop density, as well as the time and irrigation intervals and their effects on the yield of saffron are crucial. In addition, climate conditions, especially temperature, are one of the factors limiting the growth of saffron and affecting the growth pattern of the plant (Kouzegaran, 2018; Kouzegaran et al., 2014). Temperature is the main environmental factor controlling most of the physiological processes of saffron. It also regulates its developmental changes, especially flowering. The regulation of flowering of saffron by temperature and its effect on the flowering behavior has been reported in multiple studies (Koocheki et al., 2010; Molina et al., 2005, 2004). The vegetative period of saffron is around 220 days per year (Kafi, 2006). Saffron flowers appear in the fall when the weather starts to cool. The trigger for this stage is the irrigation of the saffron farm and lower temperatures. The leaves appear immediately after flowering, and in some cases, both the leaves and flowers appear from the soil simultaneously. The number and area of the leaves increase through to March and April, and then begin to decrease and reach zero in late May (Esmi, 2018). At the end of April, when the leaves are completely yellow, the plant appears to fall into a stagnant stage and fall asleep, which continues until the next year’s irrigation season. It is clear that this term is a bit misleading because during the same period cell division continues, and leaves and flowers begin to form (Molina et al., 2004). Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00008-3 © 2020 Elsevier Inc. All rights reserved.

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One of the earliest human research topics was on the effects of climate changes on agriculture. The climate of a region is influenced by a variety of internal and external factors. Some of the external factors include solar activity, western winds, and large atmospheric flows and internal factors that are dependent on the site itself such as altitude, posterior and tallness, latitudinal and distance to the sea (Kamali, 2008). Saffron can be cultivated in many regions of the world with different climates. The best climate for growing saffron is the Mediterranean climate with its warm and dry summers (Kumar et al., 2009). The geographic distribution of past and present saffron growing zones is constant (Kamali, 1989) and based on old documentation most saffron growing zones in the world are spread across the latitudes 29
42 degrees north, between Central Asia in the east to Spain in the west. These areas in Iran are in the range of 32
35 degrees latitude and are mostly at altitudes of 1000 m above sea level (Kamali, 1989). Although a lot of literature can be found on botanical aspects of saffron, not much information is available on its ecophysiological aspects. Several environmental parameters affect flower induction in saffron, of which temperature seems to play a pivotal role (Molina et al., 2005, 2004). Flower induction requires an incubation of the corms at high temperature (23 C
27 C), followed by a period of exposure at moderately low temperatures (17 C) There is evidence showing the critical importance of light and temperature in biological activities of plants including regulatory effects on dormancy period and vegetative and generative growth, particularly flowering (Halevy, 1990; Milyaeva and Azizbekova, 1978).

8.2

Climatic factors for crop production

Saffron is currently being cultivated in very diverse environmental conditions including arid countries in southeastern Europe, North Africa, and Western and Central Asia with low annual precipitation, cold winters, and hot summers (Gresta et al., 2008b; Kamali, 1989; Menia et al., 2018). Saffron is grown from 10 W to 60 E longitude and 30
42 N latitude in the Mediterranean and West Asia regions. Its cultivation regions vary from 50 m above sea level in Italy to 1630 m above sea level in Morocco (Table 8.1). The average annual rainfall in saffron cultivation areas starts from 139 mm in Iran to 700 mm in Italy (Table 8.1). In terms of temperature, the range of maximum and minimum temperatures in saffron cultivation areas is from 45 in Morocco to 214.7 C in Iran (Table 8.1). Successful production of saffron requires recognizing its ecological needs, especially climatic factors. These factors play the most important role in the expansion of saffron cultivation (Maleki et al., 2017). Agricultural production in arid and semiarid environments are largely met with inadequate water and hence saffron is considered to be a suitable crop for these areas (Azizi-Zohan et al., 2008). Climatic factors in terms of temperature and availability of water are the main factors influencing corm sprouting, flower initiation, and time of saffron flowering (Husaini, 2014).

8.2.1 Temperature The maximum and minimum tolerated temperatures for saffron are between 35 C and 40 C and 218 C to 222 C, respectively (Kouzegaran et al., 2014). The best temperature for the proper occurrence of the flowering stage of the plant is 23 C
25 C and for flowering it is around 17 C (Molina et al., 2004). The appearance of flowers in saffron is affected by factors such as radiation, temperature, nutritional elements, and water availability. However, flowering is mainly controlled by temperature. Therefore temperature is the most important indicator to estimate the plant’s flowering time. The average temperature for the onset of flowering is 12.3 C (Molina et al., 2004). The minimum temperature is the main determinant of flower formation and flower appearance, so the higher the night temperature drop, the number of flowers will be apparent the day after. It has been reported that saffron yield is significantly affected by changes in temperature and more than 70% of saffron yield variation can be designated with temperature parameters (Kouzegaran, 2018; Tosan et al., 2015). Saffron grows in many climates, but its growth is adapted in cold weather and freezing conditions in fall and winter. Although the expected appearance of its flowering time is in early fall, there is a significant variation based on region. In order to adapt to these differences, there is a need for a quantitative understanding of the flowering response of saffron to unexpected environmental changes. In many plants, reproductive growth occurs after vegetative growth, but in saffron, the flowers usually appear before the leaves and vegetative organs. Hence, known solutions for quantitative prediction of developmental stages such as growth day degree units or photothermal units are not applicable for saffron. The monthly temperature changes are considered as the most important environmental factors in the regulation of flowering of many bulbous plants (Halevy, 1990). Saffron, in contrast to many plants, has a different thermal regime and usually the onset of the plant’s activity starts with the start of the cold season. Therefore it can be considered as a coldresistant plant.

Ecophysiology of saffron Chapter | 8

121

TABLE 8.1 Some of the main saffron cultivated areas in the world and their environmental criteria. Country

Main cultivated area

Altitude (above sea level) (m)

Latitude

Longitude

Average annual rainfall (mm)

Average minimum and maximum temperature in the coldest (w) and warmest (s) month ( C)

Reference

Iran

Zaveh

1350

35 160 N

59 180 W

275

2 9.3(w) 38.8(s)

Torbat e Heydarieh

1451

35 160 N

59 130 W

262

2 9.3(w) 38.8(s)

IRIMO (2019), Kamyabi (2016)

Kashmar

1101

35 120 N

58 280 W

196

0(w) 40.1(s)

Neyshabor

1213

36 160 N

58 480 W

237

2 5.3(w) 40(s)

Tayebad

900

34 440 N

60 450 W

Ghonabad Ghaen

Italy

1056 1432



0



0



0

34 21 N 33 43 N

181

2 7(w) 42.9(s)



0

139

2 8(w) 41.7(s)



0

180

2 14.7(w) 39.5(s)



0

58 41 W 59 10 W

Ferdows

1293

34 10 N

58 10 W

148

2 9.1(w) 41.6(s)

Sarayan

1484

33 510 N

58 310 W

152.8

2 8.6(w) 41.6(s)

IRIMO (2019), NakhaieNejad et al. (2017)

Navelli

650
1100

42 140 N

13 430 E

700

2
3(w) 20
22(s)

Gresta et al. (2008b)



0



0

Sardinia in S. Gavino Monreale

50
140

39 33 N

8 48 E

300
600

10(w) 16
25(s)

Greece

Kozani Macedonia

650
700

40 180 N

21 470 E

560

2
5(w) 12.5
23(s)

Spain

La Mancha

500
600

39 240 N

3 00 W

Castille Morocco

GolkarHamzee Yazd et al. (2016), IRIMO (2019)

Taliouine

800 1200
1630



0



0

41 23 N 30 26 N

250
500

5
7(w) 16
25(s)



0

569

3
6(w) 19
26(s)



0

317

2 2(w) 45(s)

4 27 W 8 25 W

Lage and Cantrell (2009)

The optimum and suitable day temperature for corm sprouting is 23 C
25 C during the month of September, however, flowering is initiated when the average daily temperature reaches 12 C
13 C (Husaini, 2014). It seems that the beginning of saffron flowering is influenced by both the temperature and soil moisture. Gresta et al. (2009) reported that cooler environments have higher flower production, but a reduced quality of stigmas. It is well documented that the saffron flower count is positively correlated with the stigmas yield, but negatively with its unitary weight (Gresta et al., 2009).

8.2.2 Precipitation Saffron has a limited cultivation area as it is grown under narrow microclimatic conditions in the world, which are mostly located in the dry regions, with a preponderance of erratic rainfalls. Hence, it can be considered as a “niche crop” and it is a recognized as a “geographical indication” (Husaini, 2014). The growing conditions of saffron have

122

SECTION | II Safron production

become a victim of climate change, which has the potential of putting at risk the livelihood of thousands of farmers and traders (Husaini, 2014). It has been reported that precipitation has a significant effect on saffron yield from December to April compared with the other months, and for humidity the most affected months are October to February (Kouzegaran et al., 2014). Precipitation occurring in the autumn and winter seasons is very suitable after flowering of saffron, while precipitation during flowering and summer time has a negative effect on the performance of the plant (Arsalani et al., 2015).

8.3

Lifecycle

The lifecycle of saffron begins with flowering and aboveground vegetative growth at the beginning of autumn, when the plant requires rain or irrigation. The saffron growth period ends in the middle of spring after aerial parts of the plant die at about 220 days (Gresta et al., 2009). Biomass partitioning and the lifecycle of saffron have continuous stages, but the timing of the stages is to some extent different based on climatic conditions, especially temperature (Koocheki et al., 2010; Kumar et al., 2009). Saffron is propagated in a nonsexual way through corms, and they play a pivotal role in the lifecycle of saffron as they are the source of the photosynthetic plant material both after the dormant stage and in the early stages of growth. The stages of development of the saffron plant according to its physiological characteristics of the growth of its aerial parts are divided into four stages.

8.3.1 Flowering phase This is the most important stage in the plant’s growth and begins in the fall with the cool air. The flowering of saffron starts from late October to early November, with the length of this stage lasting from the start of the first flower until the last flower emerges 15
25 days later (Gresta et al., 2008b) (Fig. 8.1). Flowering, as the most important phonological stage in saffron, is directly related to the temperature of the environment (Galavi et al., 2009; Molafilabi, 2014; Molina et al., 2004).

8.3.2 Vegetative phase One of the most vulnerable stages in the growth of saffron is the vegetative stage, which starts after flowering in autumn and ends by leaves senescence in the spring. This is the longest growth stage of the plant. It starts from the end of November and lasts until late May, depending on the weather conditions of the area. At this stage, the leaves reach maturity and provide the necessary supplies for feeding the corms through photosynthesis.

8.3.3 Production of replacement corms The phonological and physiological stages of the plant are mainly carried out under the soil because unlike the seedlings, even the corm of the plant, which plays the role of the seed, is produced beneath the soil (Koocheki, 2016). In addition, the initiation and development of the flower as an economic part of the saffron take place in the soil and spends only a short part of its growth above the surface of the soil (Kafi, 2006). The developmental stages begin with

Flower number per m2

80

FIGURE 8.1 Daily flower production in Mashhad, Iran 2018.

72

70

70 60 50 40

35

33 32 26

25

30 16

20 10

1

5

14

12 7

7

6

1

0 2

4

6

8

10

12

14

16

Days from November 2

18

20

22

24

26

Ecophysiology of saffron Chapter | 8

May

Jun Period 1

Jul Period 2

Aug

Sep Period 3

123

Oct Period 4

Period 5

FIGURE 8.2 The period of the dormant phase in saffron (beginning and ending).

the formation of replacement corms and end with the completion of the replacement corms of the next generation. There are a number of meristems (buds) on each mother corm, which with their activation, the development of the replacement corms begin. The number of buds that can be converted into replacement corms has been reported to be 10 (Aghhavani-Shajari, 2017). The activity of the meristems begins after the end of the flowering season in the middle of fall (15th November to 15th December) and is divided into two parts of slow and rapid division. The slow part begins after flowering and continues until mid-March. In the second part, rapid cell division starts and continues until the leaves dry in May. At this point, the replacement corms are formed and enlarged. The number of replacement corms born from each mother corm has a reverse relationship with their size. The small replacement corms may have difficulties producing flowers in the next season. In the spring and summer, where many people believe the saffron is in its dormant stage, the development of the reproductive organs (flowers) takes place (Kafi, 2006). The growth and enlargement of replacement corms ends in the middle of May, and the mitotic activity in the cells stops in the middle of July; this period can be regarded as a complete dormant period. By August 10th, with the growth of the margins of the buds, the early leaves are formed and the veins and vasculum are created. Eventually, between the 10th and the end of August, with the widening and spreading of the upper part of the meristem area, the onset and flowering initiation are carried out in the corms.

8.3.4 Dormant phase The dormant phase begins with yellowish and leafy leaves in early May and ends with the onset of rain or irrigation with the cold air in the early autumn. The length of the course in different climate zones is on average 5 months and no specific crop operations are carried out at this stage (Kafi, 2006). This stage includes five periods: (1) entirely sleep (from late May to the first half of July); (2) the establishment and differentiation of leaves (from mid-July to late August); (3) differentiation and the initial formation of flowering organs in the bud (from late August to early September); (4) the completion of the flowering organs (until late September); and (5) the rapid growth of leaves and flower buds in the bud (from late September to the end of October) (Fig. 8.2).

8.4

Growth parameters

The trend of changes in saffron dry matter accumulation during the growing season has three stages. In the first stage, the dry matter accumulation rate is slow, up to 120 days after starting the lifecycle in fall. In the second stage the accumulation of dry matter increases more rapidly up to about 180 days after starting the lifecycle in fall (simultaneously with increasing plant growth), and in the third stage dry matter buildup increases slowly from 180 days after starting the lifecycle in the fall until the end of crop vegetative growth, which the dry matter reached its maximum amount (Esmi, 2018) (Figs. 8.3 and 8.4). In the early growing season, leaves and root systems are developed by using the mother corm’s reservoirs, but at the end of the growth cycle replacement corms are grown by translocation of reservoirs from the leaves and root (Behdani et al., 2016). The leaf, root, and corm of saffron are functionally interdependent and these three parts of the plant sustain an active balance in saffron biomass, which reflects the relative abundance of aboveground resources (leaves) compared with root and corm zone resources (Kriedemann et al., 2010). Whole-plant growth rate and other measured criteria such as mother corm, root, leaf, leaf sheath, and replacement corm weight ratios are thus an outcome of developmental stage and of environmental influences (Kriedemann et al., 2010). The fibrous roots of saffron corms are formed by the beginning of the growth season (October to November) to absorb water and nutrients, and then root dry weight increases up to the middle of the March. It then declines until the end of the reproductive growth cycle (May) (Behdani et al., 2016; Ghasemzadeh-Karimi, 2018). Ghasemzadeh-Karimi (2018) reported that root dry weight increases up to the 120 days after starting the lifecycle in the fall and then

(Dry matter (g m–2)

124

SECTION | II Safron production

3000

Mother corm weight (4.1–7 g)

2500

Mother corm weight (7.1–10 g)

2000

Mother corm weight (10.1–13 g)

FIGURE 8.3 The trend of dry matter accumulation for different mother corm sizes. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

1500 1000 500 0 60

90

120

150

180

210

Days after sowing FIGURE 8.4 The trend of dry matter accumulation for different amounts of cow manure. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

3000 –1

Cow manure (30 t ha )

(Dry matter (g m–2)

2500 Cow manure (60 t ha–1)

2000 Cow manure (90 t ha–1)

1500 1000 500 0 60

90

120

150

180

210

Days after sowing

7

14

21

Root dry weight (g m–2)

16 14 12

FIGURE 8.5 The trend of root dry weight of saffron for different irrigation intervals days after sowing. From Ghasemzadeh-Karimi, J., 2018. Study the growth indices and corm characteristics of saffron affected by different irrigation regimes and organic fertilizers (MSc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

10 8 6 4 60

90

120 Days after sowing

150

180

decreases (Figs. 8.5 and 8.6). In another study on saffron, the maximum root weight was shown at 90 days after first irrigation in the autumn and it continued with a declining trend (Fallahi and Mahmoodi, 2018). The roots of saffron corms are active until mid-February and play a nutritional role for the plant. From then on the roots do not play a role, due to the demolition of the mother corms. Increasing of the replacement corms weight perhaps is then related to the transmission of the mother corm resources and also photosynthesis of leaves. Therefore any enhancement of soil nutrient content should be provided before late November to early January (Molafilabi, 2014). Some research shows that the source of fertilizer and irrigation can have an effect on root development in saffron (Ghasemzadeh-Karimi, 2018; Shariatmadari, 2018). Ghasemzadeh-Karimi (2018) stated that cow manure and urban compost fertilizer treatments increased the dry weight of saffron root compared with other fertilizer treatments (Fig. 8.6).

Ecophysiology of saffron Chapter | 8

Urban compost Hen manure Urea fertilizer

Vermicompost Cow manure Control

20 Root dry weight (g m–2)

18 16 14

125

FIGURE 8.6 The trend of root dry weight of saffron for different fertilizer sources days after sowing. From Ghasemzadeh-Karimi, J., 2018. Study the growth indices and corm characteristics of saffron affected by different irrigation regimes and organic fertilizers (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

12 10 8 6 4 2 60

90

120

150

180

Days after sowing M.C.

(Dry matter (g m–2)

1225.00

R.

L.

FIGURE 8.7 The trend of mother corm (M.C.), root (R.), leaf blade 1 leaf sheath (L.), and replacement corm (R.C.) dry weight of saffron days after flowering. From Shariatmadari, Z., 2018. Physiological and morphological study of saffron corm and flower in response to different irrigation frequency, corm size, organic and NPK fertilizers (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

R.C.

1025.00 825.00 625.00 425.00 225.00 25.00 30

60

90 120 Days after flowering

150

180

The maximum amount of dry matter accumulation during the growing season depends on irrigation and the amount of sowing corms, sowing depth, and corm size in the first year (Behdani et al., 2016; Esmi, 2018; GhasemzadehKarimi, 2018; Mirhashemi et al., 2015; Moallem-Banhangi, 2016; Razavian, 2017; Shariatmadari, 2018). It has been reported that the leaf dry weight increases up to 150 days after flowering and then decrease more rapidly when it finally reached zero in midspring (Behdani et al., 2016; Renau-Morata et al., 2012; Shariatmadari, 2018) (Fig. 8.7). Fallahi and Mahmoodi (2018) stated that leave weight reaches its maximum up to 114 days after the first irrigation and then decreased. Renau-Morata et al. (2012) reported that the share of saffron leaves is almost 90% of the biomass accumulation and the remaining B10% biomass is derived from the mother corm. The dry weight of replacement corms begins to increase at the beginning of the vegetative growth period, so that the trend of the increasing is high at the beginning of the growing season, during 150
180 days after flowering, the weight gain of replacement corms will be linearly increased (Fig. 8.7). So at the end of the growing season, the dry weight of the replacement corms is fixed. It has been reported that the weight of replacement corms increases from November to May (Behdani et al., 2016; Feizi et al., 2015; Renau-Morata et al., 2012) (Fig. 8.7). Saffron mother corms provide resource needs to produce roots, flowers, and leaves at the beginning of the growing season (Behdani et al., 2016). At the same time as the replacement corms are starting to form and the weight is increasing, the mother corm weights are linearly reduced and at the end of the growth season, mother corms completely disappear (Fig. 8.7).

8.4.1 Leaf area index The leaf area index (LAI) is a dimensionless criterion defined as one-sided surface area of leaf per unit ground surface area (Breda, 2003). The leaf is the main and active component of saffron plant photosynthesis, so its efficiency in absorbing and using solar energy determines the production and yield of the saffron. Therefore the leaf area of the saffron is a key determinant of radiation absorbance, photosynthesis, and the exchange of energy and water (Yin et al., 2019).

126

SECTION | II Safron production

TABLE 8.2 The maximum leaf area index (LAI), crop growth rate (CGR), and total dry matter (TDM) in the growing period of saffron in the first year. Maternal corm weight (g)

LAI

4.1
7

7.1
10

10.1
13

0.365

0.456

0.521

Cow manure (tons ha21) 30 0.406

60 0.450

90 0.486

Irrigation method Flooding irrigation 0.401

Sprinkler irrigation 0.494

Drip irrigation 0.448

CGR (g m22 day21)

13.37

16.35

19.12

15.04

15.35

18.46

14.30

18.76

15.78

TDM (g m22)

1402




2281

1760

1889

1985

1633

2062

1938

From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

According to the unique phenology of saffron, the leaves appear in the middle of autumn after flowering, and its number and surface increase to reach the maximum leaf area in March to April, then with drying of some leaves, leaf area decrease and it reaches zero by late May (Kafi, 2006). As the age of saffron increases, so does the leaf area. The leaf area depends on corm size, number of meristems per corms, number of corms per clone, water availability, soil nutrient, and other environmental factors. In the first year of new sowing corms, there is only one mother corm with 2
5 meristems per corm in each clone. Thus as the age of the farm increases, the number of corms will increase and consequently the leaf area will increase. The LAI, light extinction coefficient, and radiation use efficiency are important ecophysiological characteristics for understanding of saffron growth, development, and radiation absorption (Mirhashemi et al., 2015). A few studies have been done to determine the LAI of saffron, indicating that its LAI is low and did not exceed 1.8 in optimal conditions (Esmi, 2018; Ghasemzadeh-Karimi, 2018; Mirhashemi et al., 2015; Moallem-Banhangi, 2016; Razavian, 2017; Shariatmadari, 2018). By increasing the age of saffron from 1 to 2 years, the maximum LAI of saffron will increase from 0.33 to1.81 (Mirhashemi et al., 2015). The results of Esmi (2018) showed that by increasing mother corm weight up to 13 g (4
13 g), cow manure up to 90 tons ha21 (30
90 tons ha21), and improving the irrigation system (drip and sprinkler irrigation) the maximum LAI of saffron 210 days after planting were 0.521, 0.466, and 0.494, respectively (Table 8.2). The photosynthetic capacity of leaves remains nearly constant during the saffron growth period (Renau-Morata et al., 2012) and contributed to 87%
91% of the biomass accumulation of the whole plant. Saffron LAI has a nonlinear trend and followed a sigmoid function. At the end of the flowering stage, the LAI initially begins to increase with a linear trend until 210 days after starting the lifecycle in the fall and then leaves dies and it reached zero. The maximum rate of LAI increases between 90
180 days after starting the lifecycle in fall, which is simultaneous with the maximum vegetative growth rate of saffron (Figs. 8.8
8.11).

8.4.2 Crop growth rate The total dry matter production of a plant community per unit of land area per day is called the crop growth rate (CGR). The CGR of saffron slightly decreases at the beginning of the vegetative growth period (120 days after starting the lifecycle in autumn) probably due to higher use of mother corm reserves and then take an additive trend from 120 days to about 180 days after starting the lifecycle in the fall, and finally, at the end of the vegetative growth period, take a decreasing trend (Esmi, 2018; Ghasemzadeh-Karimi, 2018; Moallem-Banhangi, 2016; Razavian, 2017; Shariatmadari, 2018) (Figs. 8.12
8.14). The maximum reported CGR of saffron vary from 1.73 to 19.2 (g m22 day21), depending on many factors such as the time of starting the lifecycle in fall, sowing depth, corm size, and the number of sowing corms in the first year, irrigation, and soil fertility management (Esmi, 2018; Ghasemzadeh-Karimi, 2018; Moallem-Banhangi, 2016; Razavian, 2017; Shariatmadari, 2018).

8.4.3 Relative growth rate The relative growth rate (RGR) is the most useful and widely used growth analysis index, derived from the growth of cell populations with unrestricted resources, which is where light, space, and nutrient supply are not limited

Ecophysiology of saffron Chapter | 8

FIGURE 8.8 The trend of leaf area index for different mother corm sizes in the first year. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

0.7 Mother corm weight (4.1–7 g)

0.6

Mother corm weight (7.1–10 g)

Leaf area index

0.5

127

Mother corm weight (10.1–13 g)

0.4 0.3 0.2 0.1 0.0 60

90

120

150

180

210

Days after sowing

0.7

Cow manure (30 t ha–1)

0.6

Cow manure (60 t ha–1) Cow manure (90 t ha–1)

0.5 Leaf area index

FIGURE 8.9 The trend of leaf area index for different amounts of cow manure in the first year. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

0.4 0.3 0.2 0.1 0.0 60

90

120 150 Days after sowing

180

210

FIGURE 8.10 The trend of leaf area index for different irrigation intervals in the second year. From Ghasemzadeh-Karimi, J., 2018. Study the growth indices and corm characteristics of saffron affected by different irrigation regimes and organic fertilizers (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

0.25 Irrigation interval 7 days Irrigation interval 14 days

Leaf area index

0.2

Irrigation interval 21 days

0.15 0.1 0.05 0

30

60

90 Days after flowering

120

150

Leaf area index

128

SECTION | II Safron production

0.7

10 cm

0.6

15 cm

FIGURE 8.11 The trend of leaf area index for different sowing depths of saffron corms. From Razavian, M., 2017. The effects of corm weight and depth of planting on growth characteristics and yield of flower and corm of saffron (Crocus sativus L.) (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

20 cm

0.5 0.4 0.3 0.2 0.1 0.0 60

90

120

150

180

210

Crop growth rate (g m–2 day)

Days after sowing

30

Mother corm weight (4.1–7 g)

25

Mother corm weight (7.1–10 g)

20

FIGURE 8.12 The trend of crop growth rate for different mother corm sizes. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

Mother corm weight (10.1–13 g)

15 10 5 0 60

90

120

150

180

210

–5 –10

Days after sowing

30 25 Crop growth rate (g m–2 day)

FIGURE 8.13 The trend of crop growth rate for the different amounts of cow manure. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

Cow manure (30 t ha–1) –1 Cow manure (60 t ha )

20

–1 Cow manure (90 t ha )

15 10 5 0 60

90

120

150

–5 –10

Days after sowing

180

210

Ecophysiology of saffron Chapter | 8

129

Crop growth rate (g m–2 day)

4 Irrigation interval 7 days

3.5

Irrigation interval 14 days

3 Irrigation interval 21 days

2.5 2 1.5 1 0.5 0 30

60

90

120

150

180

Days after flowering FIGURE 8.14 The trend of crop growth rate for different irrigation intervals in the second year. From Ghasemzadeh-Karimi, J., 2018. Study the growth indices and corm characteristics of saffron affected by different irrigation regimes and organic fertilizers (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

0.005 Cow manure (30 t ha–1) Cow manure (60 t ha–1)

Relative growth rate (g g day)

0.004

Cow manure (90 t ha–1)

0.003

0.002

0.001

0 60

90

120

150

180

210

–0.001 Days after sowing FIGURE 8.15 The trend of relative growth rate of saffron for different manure quantities days after sowing. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

(Kriedemann et al., 2010). The RGR can be stated in terms of differential calculus as RGR 5 (1/W) (dW/dt) so that RGR is increasing in dry mass (dW) per increase in time (dt) divided by existing biomass (W). Studies show that saffron RGR declines slightly from 90 to 120 days after planting, simultaneously with the onset of growth of leaves and replacement corms. At this stage, the existing leaf area is still low and due to the low ratio of photosynthetic tissues to respiration, the RGR is slightly reduced (Esmi, 2018; Moallem-Banhangi, 2016; Razavian, 2017) (Figs. 8.15
8.17). In the second stage (the time interval from 120 to 180 days after planting), with the increase in vegetative growth rate, the RGR increases due to the faster growth of the leaves and the maximum number of young tissues involved in photosynthesis and reaches its maximum. In the third stage, 180 days after sowing the RGR decreases linearly. It seems that although the amount of dry matter of the plant increases with time, due to the aging of the leaves, photosynthesis decreases relatively. Finally, 240 days after planting, the RGR is zero or even negative (Figs. 8.15
8.17). The reason for this can be attributed to the fact that at the end of the growth season, not only does the dry weight of the saffron plant not increase, but also because the plant respiration rate is more than the amount of photosynthesis of the old and young leaves of the plant, the relative growth rate is negative

130

SECTION | II Safron production

Relative growth rate (g g day)

0.005

Leakage irrigation Drip irrigation

0.004

Sprinkler irrigation

0.003 0.002 0.001 0 90

120

150

180

210

240

–0.001 –0.002

Days after sowing

FIGURE 8.16 The trend of relative growth rate of saffron for different irrigation methods days after sowing. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

Relative growth rate (g g day)

0.04 0.02 0 30

60

90

120

150

180

–0.02 Irrigation interval 7 days

–0.04 Irrigation interval 14 days

–0.06 Irrigation interval 21 days

–0.08 –0.1 Days after flowering

FIGURE 8.17 The trend of relative growth rate of saffron for different irrigation intervals in the second year. From Ghasemzadeh-Karimi, J., 2018. Study the growth indices and corm characteristics of saffron affected by different irrigation regimes and organic fertilizers (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

(Esmi, 2018; Moallem-Banhangi, 2016; Razavian, 2017; Shariatmadari, 2018). It is also due to the fact that the parts added to the plant are structural tissue and are not active metabolic tissue and thus do not contribute to the growth and cause a decrease in RGR.

8.4.4 Net assimilate rate The net assimilate rate (NAR) shows a plant’s net photosynthetic efficiency in catching the light, assimilating CO2, and storing photosynthetic components. Variation in NAR can originate from differences in canopy architecture and light interception, photosynthetic activity of leaves, respiration, and transport of photosynthetic components and storage capacity of sinks (Kriedemann et al., 2010). Studies have shown that the trend of changes of saffron NAR is similar in different treatments such as different animal manure application rates or different irrigation methods. However, 90
120 days after planting, the rate of NAR had a decreasing trend, due to being at the vegetative growth stage, when the leaves are being developed (Esmi, 2018; Moallem-Banhangi, 2016; Razavian, 2017) (Figs. 8.18 and 8.19). It seems in this stage the rate of respiration of plant tissues is much higher than the rate of photosynthesis in the saffron leaves. The trend of NAR increases from 120 up 180 days after planting when the leaves are highly developed and photosynthesis is increased. At this stage, due to the fact that the amount of plant photosynthesis is much higher than the rate of respiration of saffron tissues, the NAR increases with a very sharp gradient and reaches its maximum. In the next step, and at 180
210 days after planting, the rate of NAR decreases due to increasing respiration in plant tissues and eventually the rate of NAR reaches zero (Esmi, 2018; Shariatmadari, 2018).

Ecophysiology of saffron Chapter | 8

0.004 Cow manure (60 t ha–1)

–1 Cow manure (30 t ha )

Cow manure (90 t ha–1)

Net assimilate rate (g m–2 day)

0.003

131

FIGURE 8.18 The trend of net assimilate rate of saffron for different manure quantities days after sowing. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

0.002

0.001

0 60

90

120

150

180

210

–0.001 Days after sowing

0.004

Leakage irrigation

Drip irrigation

Sprinkler irrigation

Net assimilate rate (g m–2 day)

0.003

FIGURE 8.19 The trend of net assimilate rate of saffron for different irrigation methods days after sowing. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

0.002

0.001

0 60

90

120

150

180

210

–0.001

Days after flowering

8.4.5 Leaf area ratio The leaf area ratio (LAR) represents the ratio of leaf area (photosynthetic tissue) to dry weight of the whole plant (respiratory tissue). It shows the quantity of the leaves present on a plant and is an indicator of the efficiency where a plant deploys its photosynthetic resources. Generally, the maximum saffron LAR appears at the beginning of the vegetative growth stage, which can be differed by sowing depth and amount of corms as seed per square meter (MoallemBanhangi, 2016) (Figs. 8.20 and 8.21). The LAR rapidly reduces at the end of the growing season. It seems that due to the rapid growth of the replacement corms, which much of the photosynthesis products are allocated to the underground parts of the saffron plant, the LAR declines from the early stages of growth. This suggests that early vegetative growth occurs at the same time as the growth of the leaves, the growth of the underground organs begins at a rapid pace.

8.4.6 Leaf weight ratio The leaf weight ratio (LWR) is stated as the dry weight of leaves to the whole-plant dry weight (a measure of biomass allocation to leaves) and is expressed in g g21. Based on the study by Shariatmadari (2018), the trend of LWR changes follows an incremental process up to 150 days after flowering and then drastically declines (Fig. 8.22). As Fig. 8.22 shows, at the

FIGURE 8.20 The trend of saffron leaf area ratio for different depths. From Moallem-Banhangi, F., 2016. The effects of different quantity and planting depths of corms on flower and corm yield of saffron (Crocus sativus L.) (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

30 10 cm

Leaf area ratio (cm g–1)

25 15 cm

20 20 cm

15 10 5 0 90

120

150 Days after sowing

180

210

FIGURE 8.21 The trend of saffron leaf area ratio for different amounts of corm as seed. From Moallem-Banhangi, F., 2016. The effects of different quantity and planting depths of corms on flower and corm yield of saffron (Crocus sativus L.) (M. Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

30 7 t ha

8 t ha–1

25

Leaf area ratio (cm g–1)

–1

9 t ha–1 10 t ha–1

20 15 10 5 0 90

120

150 Days after sowing

180

210

FIGURE 8.22 The trend of saffron mother corm, root, leaf, and replacement corm weight ratios in days after flowering. From Shariatmadari, Z., 2018. Physiological and morphological study of saffron corm and flower in response to different irrigation frequency, corm size, organic and NPK fertilizers (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

1.20 Mother corm weight ratio

1.00

Root weight ratio

Leaf weight ratio

Ratio (g g–1)

0.80

Replacement corm weight ratio

0.60

0.40

0.20

0.00 30

60

90

120

Days after flowering

150

180

Ecophysiology of saffron Chapter | 8

2500

FIGURE 8.23 The trend of decreasing mother corm reserves and increasing replacement corm reserves. From Esmi, R., 2018. Physiological and morphological study of saffron corm and flower in response to irrigation methods, different corm size and cow manure (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

Replacement corm weight (4.1–7 g) Replacement corm weight (7.1–10 g)

2000

Replacement corm weight (10.1–13 g)

Mother corms

Dry matter (g m–2)

Mother corm weight (4.1–7 g) Mother corm weight (7.1–10 g)

1500

133

Mother corm weight (10.1–13 g)

1000

500

0

0

30

60

90 120 Days after planting

150

180

210

peak of the LWR, the replacement corm weight ratio (RCWR) was 0.38 g g21, while with the declining LWR and root weight ratio (RWR) the RCWR rapidly increased and at the end of the growing season reached its maximum. This indicates that the remobilization of resources takes place, in which biomass is translocated from leaves and roots to replacement corms at the end of the growth cycle (Fallahi and Mahmoodi, 2018). However, the trend of RCWR increases up to 90 days after flowering, then stabilizes until 150 days after flowering, at which point it increase sharply again.

8.4.7 Corms Leaves and mother corms contribute biomass for continuing the vegetative development of saffron, which varies throughout the growing season. In the early period of vegetative growth after flowering, during which the roots and leaves develop, the majority of the remaining reserves of the mother corms are mobilized and maintain the vegetative growth (Renau-Morata et al., 2012). Generally, in the first 2 months after sowing, replacement corms have no growth, and their growth initiation coincides with the end of the flowering season. The following growth of the replacement corms (the key sinks of the plant) is mainly supported by the photosynthesis in the leaves, which contributes 90% of the biomass accumulation in the whole plant (Renau-Morata et al., 2012). The photosynthetic rate is constantly very high during vegetative growth but is reduced in bigger corms (Renau-Morata et al., 2012). The dry weight of the replacement corms exponentially begins to increase with the beginning of the vegetative growth period while at the end of the growing season, their dry weight is fixed. Simultaneous with the increase in replacement corm weight, mother corm weight is linearly reduced, and at the end of the growth season, they fully disappear (Fig. 8.23) (Esmi, 2018). Replacement corm production is increased with increasing number of irrigations as a result of the direct connection between photosynthesis and soil water potential (Koocheki et al., 2019; Renau-Morata et al., 2012).

8.4.8 Whole plant The growth trends of different saffron organs during the lifecycle shows that the critical stage for biomass partitioning is 120
150 days after flowering (Behdani et al., 2016; Esmi, 2018; Ghasemzadeh-Karimi, 2018; Moallem-Banhangi, 2016; Razavian, 2017; Shariatmadari, 2018). Biomass partitioning showed two separate stages before and after this critical partitioning stage. During the first stage, the biomass is mainly assigned to leaves and roots, whereas during the second stage replacement corms receive the main share of the dry matter (Behdani et al., 2016). Consequently, the mother corm dry weight have a declining trend during the 3
6 months after the first irrigation (Behdani et al., 2016; Esmi, 2018; Ghasemzadeh-Karimi, 2018; Moallem-Banhangi, 2016; Razavian, 2017; Shariatmadari, 2018). However, the root, leaf, and replacement corm dry weights increase during the 3
6 months after the first irrigation. In another study on saffron, the maximum leaf area surface was obtained in 90
150 days after irrigation, which coincides with the mobilization of most parts of the mother corm reservoirs (Moallem-Banhangi, 2016). The growth of replacement corms mainly depend on saffron leaf photosynthesis. Therefore the appropriate development of leaves has a significant effect on the production of larger replacement corms and thereby on the yield of saffron in the coming year (Behdani et al., 2016).

134

SECTION | II Safron production

After flowering during saffron root and leaf development, most of the mother corm reserves are depleted. The growth of replacement corms initiates when the roots and leaves reach their maximum size, and this development mainly depends on leaves photosynthesis (Renau-Morata et al., 2012). Saffron mother corm reserves only contribute 10% to the biomass during vegetative development. The photosynthetic rate is constantly high (26 mol m22 s21) during the growing period but is reduced in plants from the largest corms (Renau-Morata et al., 2012). A sink capacity limitation increases during growth of replacement corms since the increase of biomass is limited before total leaf senescence. A decrease in the photosynthetic rate is observed under water stress, but the supply of water to the leaves from the corms and roots is found to maintain a still high photosynthetic rate (12 mol m22 s21) even at highly negative soil water potentials (Renau-Morata et al., 2012).

8.4.9 Source and sink relationship in the growth organs Understanding the source
sink relations in terms of distribution of nutrients and assimilates in different organs and tissues during different phenological stages of crops is the main topic of crop physiology (Behdani et al., 2016; Bennett et al., 2012). Saffron leaves are fixing carbon during photosynthesis. Fixed carbon is either used for the cell’s respiration and growth, or it is transferred mainly in the form of sucrose to other organs to support their growth and development (Chen, 2005; Turgeon, 1989). Therefore organs in any plant can be mostly divided into source and sink organs. Mature leaves are represented as source organs in saffron, which are usually defined as exporters of fixed carbon, while mainly replacement corms are represented as sink organs that are referred to as importers of assimilating (Turgeon, 1989). The storage sinks in saffron (replacement corms) are specialized as reproduction organs, which continuous the lifecycle of the plant in the next growing season (Sonnewald and Willmitzer, 1992). Although replacement corms are storage sinks, during sprouting in the next growing season they turn into source organs where the stored compounds are used as a source of energy to provide nutrients for the growth of the buds (Sonnewald et al., 1997). The main strategy in saffron in terms of the evolution of sink and source is to increase sink strength, which has been considered as a product of sink size and sink activity (Ho, 1988; Sonnewald and Willmitzer, 1992; Turgeon, 1989). Fallahi and Mahmoodi (2018) believe that the main obstacle in terms of increasing the corm size of saffron (sink) is a source limitation. In other words, low LAI of saffron (0.1
0.3), poor soil nutrients, and harsh climate conditions are the main reasons for source limitation in most of saffron growing areas.

8.5

Effects of environmental changes on the quality of saffron

The quality of saffron is determined by the three metabolites of crocin, picocrocin, and safranal. Its aroma is related to escaping essential oils so that 70% of saffron escape rate is safronal (Kyriakoudi et al., 2015). This material is obtained after decomposition of the initial glycosides. Geographical factors including climatic, edaphic, physiographic, and biotic factors are the most important issues and may affect the amount of saffron stigma glycosides, especially safranal levels. Saffron has considerable potential as a crop for the Mediterranean environment, where the saffron is able to escape unfavorable conditions of high temperature and low rainfall by means of autumn flowering. Moreover, in the Mediterranean environment, very-high-quality saffron spice can be produced (Gresta et al., 2008a). Macchia et al. (2013) reported that the place of cultivation in terms of climate and soil types and dehydrating time period of stigmas after harvesting were the main factors of differences in safranal, isophorone, 4-keto isophorone, c-pyronene, and dihydrooxophorone in saffron stigmas. Lage et al. (2010) and Lage and Cantrell (2009) analyzed the environmental impact on saffron quality under different Moroccan environments and stated that the altitude significantly affects crocins content (R2 5 0.84). The results of Caser et al. (2019) and Giorgi et al. (2017) revealed that the Alpine area in Italy produces the best quality of saffron in terms of crocin, picocrocin, and safranal contents. Giorgi et al. (2017) suggested the reason for such a result could be due to harvesting the flowers during the first hours of the day while blossoms are still closed and light is not degrading the crocins that are inside the stigmas, drying process and storage. Gresta et al. (2008a) noted that saffron quality according to the apocarotenoid content (ISO rules) was strictly related to sowing time. This result shows that environmental conditions play an important role in the buildup of secondary metabolites (apocarotenoids), which is responsible for saffron stigma quality. It has been reported that the use of arbuscular mycorrhizal symbionts as biostimulants enhanced the antioxidant activity and the content of bioactive compounds of saffron such as picrocrocin, crocin II, and quercitrin (Caser et al., 2019). Studies show that there is a negative correlation between safranal contents and potassium and electrical conductivity of the soil (Ershad et al., 2014).

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Based on the results of Molafilabi (2014) and Poggi et al. (2010), the production of saffron under controlled environment was superior in quality for crocin, picocrocin, and safranal to that obtained in the field. This could be due to the provision of optimal control of the climate and nutritional conditions of saffron, which can be used in hydroponic substrates to provide the necessary nutrition to the plant by spraying and feeding the leaves (Molafilabi, 2014).

8.6

Yield determination

Saffron flower and corm yield depend on many factors such as climatic, edaphic, agronomic, and to some extent physiographical factors. It has been documented that climatic and edaphic factors are the main factors influencing the flower and corm yield of saffron. However, climatic factors such as temperature and precipitation are the main reasons to limit saffron cultivation areas to particular geographical regions in the world. There is a linear relationship between minimum and maximum temperature and precipitation of the cultivated area with the saffron yield (Kouzegaran, 2018). Based on the output of the different climate change models it has been predicted that the average temperature will increase in short, medium, and long periods of time in the main saffron production areas of the world (Khorasan Razavi and southern Khorasan, Iran) (Kouzegaran, 2018). Saffron yield will decrease by increasing temperature and decreasing precipitation up to 10% in the short term (2025
50), 20% in medium term (2051
75), and up to 33% in the long term (2076
2100) (Kouzegaran, 2018). Sowing date, sowing depth, corm size, number of sowing corms in the first year, irrigation, and corm storage are the most important issues for increasing saffron yield in the first and subsequent years from an agronomical factor point of view. The optimal sowing depth, while providing optimum conditions for flower appearance and plant emergence, is also effective in protecting corms from chilling and freezing stress in the winter and also heat and drought in the summer. Saffron is an annual plant from a botanical point of view, but in farming systems it is actually a perennial plant (Koocheki, 2016). It can be sown each year like other annual plants. There have been many studies focused on corm density in the first year of saffron sowing. It has been reported that the maximum flowers and corms yield are obtained at 200 corms per square meter (Koocheki et al., 2014). The rate of saffron yield increase, however, decreases with the age of the saffron field because of the overcrowding of new corms, smaller corm size, and subsequent reduction in the number of flowers produced per corm (Behnia et al, 1999). Good agricultural practices including sowing appropriate corm size ( . 8 g), proper inter- and intrarow spacing (10 3 20 cm), using organic and biological fertilizers such as animal manure, vermicompost and biofertilizers, and water scheduling during critical stages (sprouting to flowering) guarantees high factor productivity and high income per unit area (Koocheki et al., 2019, 2014; Yasmin and Nehvi, 2013).

8.7

Conclusion

Better understanding of the roles the major factors responsible for higher production and productivity of saffron play can help producers manage their land and provide suitable conditions for producing higher quality and quantity of saffron. These factors can be summarized as better understanding of the impact of climate change (mainly increasing temperature and declining precipitation, which have negative impacts) on saffron production and productivity; planting larger corms and using higher planting density for increasing growth indices; choosing proper corm sowing depth for protecting corms against freezing and heat stresses during winter and summer, respectively; and proper nutrient management with adequate irrigation facilities.

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216 (in Persian). Moallem-Banhangi, F., 2016. The effects of different quantity and planting depths of corms on flower and corm yield of saffron (Crocus sativus L.) (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian). Molafilabi, A., 2014. Effect of new cropping technologies on growth characteristics, yield, yield components of flower and corm criteria of saffron (Crocus sativus L.) (Ph.D. thesis). Ferdowsi University of Mashhad, Mashhad, Iran (in Persian). Molina, R.V., Garcia-Luis, A., Coll, V., Ferrer, C., Valero, M., Navarro, Y., et al., 2004. Flower formation in the saffron crocus (Crocus sativus L.). The role of temperature. Acta Hortic. 650, 39
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Chapter 9

Advances in modeling saffron growth and development at different scales Mehdi Nassiri Mahallati Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 9.1 Introduction 9.2 Statistical models 9.2.1 The basics 9.2.2 Crop-weather models for saffron yield prediction 9.3 Artificial neural networks 9.3.1 Artificial neural networks, an overview 9.3.2 Application of artificial neural networks for saffron 9.4 Application of response surface modeling in saffron production 9.4.1 Statistical background 9.4.2 Central composite designs 9.4.3 Application 9.5 Dynamic simulation models

9.1

139 140 140 140 143 143 144 146 146 146 147 148

9.5.1 Advances in development of simulation models for saffron 9.5.2 Radiation-based model for saffron growth 9.6 Modeling saffron development and flowering 9.6.1 Hypothetical model of saffron development 9.6.2 Developmental responses in crop species 9.6.3 Structure of the model 9.6.4 Simulation of saffron response to climate change 9.7 Land suitability and zoning methodology for saffron 9.7.1 Objectives and methods of zoning 9.7.2 Application of zoning schemes to saffron 9.8 Conclusion References Further reading

150 152 154 154 155 155 158 161 161 162 163 164 167

Introduction

Crop models are sets of mathematical equations that represent processes within a predefined plant system as well as the interactions between crops and the environment. Considering the complexity of agricultural systems and the existing gaps in present knowledge, it seems impossible to express entire processes of a crop system in mathematical terms provided that agricultural models are still simplified versions of reality (Wallach et al., 2014). The ultimate purpose of developing crop models is to get a precise estimation of the economic yield. However, depending on the availability of information and data at the interested scale, crop models are developed at different levels of complexity (Jones et al., 2017). Therefore they may range from multivariate regression, so-called empirical models based on monthly weather variables intended to predict crop yields at regional scale (Paswan and Ara Begum, 2013), to process-based ones, so-called mechanistic models of plant growth, developed for getting insight into the crop physiological interactions (Janssen et al., 2017). Since 1970, several mathematical models at different levels of sophistication have been developed for simulation of growth, development, and yield of cultivated crops. These models are extensively used for different purposes such as crop management, yield gap analysis, crop-pest interactions, and climate change impact studies (Jin et al., 2018; Jones et al., 2017; Ritchie and Alagarswamy, 2002; Van Ittersum et al., 2013). However, current modeling efforts are mainly focused on globally important crops (e.g., wheat, rice, corn, soybean) and underutilized species grown locally and mainly in developing countries are ignored. This could be to some extent due to lack of experimental information about these crop species and poor availability of long-term weather and soil data at the local level, which are the main inputs of any crop model (Kandiannan et al., 2002). Obviously simulation models of locally important crops such as saffron should be developed by local researchers in the producing countries, but so far this important area of agronomic sciences has been overlooked. Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00009-5 © 2020 Elsevier Inc. All rights reserved.

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This chapter aims to present methods to integrate the existing knowledge about saffron (Crocus sativus L.) growth for development of mathematical models at different levels of complexity for the prediction of yield and growth processes (e.g., flower emergence or vegetative growth). In each section a brief description of the underlying topic is presented, followed by some applications on saffron. Furthermore, application of modeling approaches in regional studies on issues such as climate change impacts and agroecological zoning of saffron are also discussed.

9.2

Statistical models

9.2.1 The basics Time series analysis of long-term yield data represents three sources for interannual variation of crop yields including trends, direct weather effects, and indirect weather effects (e.g., pests, diseases, weed competition) (Gommes, 1999). Statistical models also known as crop-weather models are multivariate regression equations that describe the relation between crop yield and one or more weather variables. The main advantage of regression models is that they need a limited number of data and yield could be satisfactorily predicted by using monthly values of weather variables (Gommes, 2001). In addition, mathematical calculations can be readily conducted using any standard statistical software or even in a spreadsheet. Due to their structural simplicity and limited data requirements, regression models are well known as one of the principle methods for quantitative analysis of crop-weather relationships especially when the objective is yield prediction at large spatial scales (e.g., districts, provinces, regions) (McKeown et al., 2006). The general approach is to develop a function between dependent variable (crop yield) and independent variable(s) consisting of selected meteorological variables. The statistical procedure by which the coefficients of a regression model are estimated is known as calibration (Gommes et al., 2010). While the linear relationship is the first assumption for describing the dependence of crop yield on weather, cropweather models cannot always be expressed by linear functions. When there are good reasons that the response of crop yield to a given weather variable is not linear, a quadratic term in addition to the linear term could be introduced into the regression equation (Lobell et al., 2007; Schlenker and Roberts, 2009). For example, the relationship between precipitation and yield is quadratic because precipitation could be too high or low for maximum yield, but an optimal amount exists between extreme dryness and wetness. Similar to precipitation, the relationship between temperature and crop yield is be quadratic with an optimal point located between the lowest and the highest temperatures. The main shortcoming of statistical models is their poor performance outside the range of values for which they have been calibrated, provided that regression models are site-specific; extrapolation of their results should be avoided to avoid the possibility of considerable prediction error (Gommes, 2001). Since the pioneer work of Smith (1914) regression models have been extensively used for yield prediction of different crop species at large spatial scales (Kandiannan et al., 2002; Lobell et al., 2007; Mathieu and Aires, 2016; Qian et al., 2009; Tannura et al., 2008). More details on statistical aspects of crop-weather models including parameter estimation and validation methods could be found in related literature (e.g., Baier, 1977; Gommes et al., 2010; McKeown et al., 2006).

9.2.2 Crop-weather models for saffron yield prediction While regression models for saffron yield prediction have not been reported by other saffron-producing countries, several crop-weather models have been developed to predict saffron yield in the main production regions of Iran (Hosseini et al., 2008; Kouzegaran et al., 2018; Sanaeinejad and Hosseini, 2009; Salari et al., 2017; Tosan et al., 2015). Despite the difference in the length and period of time series, the selected meteorological variables, and statistical methods applied in theses studies, the overall results indicates that weather variables account for 33%
85% of the observed saffron yield variation and monthly temperature is the best yield predictor compared to other variables when saffron yield prediction at large scales is the objective. However, the best combination of variables reported for yield prediction was to some extent different in these studies. For example, Hosseini et al. (2008) found that the minimum temperatures of April, May, and June are the best predictors of saffron yield at the district level but Sanaeinejad and Hosseini (2009) showed that the model had better performance when both the maximum and minimum temperatures of April and May were introduced in the regression equation. Tosan et al. (2015) reported that the range (maximum minus minimum) of April and May temperatures can also be considered as input variables for satisfactory prediction of saffron yield. Kouzegaran et al. (2018) used extreme weather events as input variables in their regression model. They first

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calculated the extreme values of monthly and seasonal temperature (27 extreme values) and precipitation (11 extreme values) for a 25-year period based on the method recommended by the Expert Team on Climate Change Detection and Monitoring (ETCCDM) of the World Meteorological Organization. The regression model with 38 independent variables was then subjected to stepwise selection. The results indicated that saffron yield was precisely predicted (R2 5 0.88) with eight extreme weather variables. Nonetheless, for development of reasonable crop-weather models some statistical considerations should be satisfied, which were not fully employed in the above-mentioned studies. The observed variation in crop yields in addition to weather effects represents a trend, which is due to technological improvements (irrigation, fertilizers, and other management practices). Trend is independent of meteorological variability and therefore time series of crop yields should be detrended before further statistical analysis (Gommes, 2001). In fact, regression models describe the relationships between weather variables and detrended yield. Detrending of yield data (i.e., removing trend from actual yield) is mathematically simple and would be possible by subtracting the estimated yield for each year in the time series from the actual yield of the corresponding year. However, it was only adopted by Hosseini et al. (2008) who studied the relation between saffron yield and weather variables (monthly precipitation and minimum, maximum, and average temperatures) for periods during 1985
2005 in six districts. In Fig. 9.1 original and detrended saffron yields for a selected district are shown where it can be seen that detrended yield (Fig. 9.1A) representing weather effects is still considerably high and could be used as a dependent variable in the crop-weather model. On the other hand, when there is a significant trend in the weather variables detrending the time series is probably not a solution and it is recommended to use the actual yield as the dependent variable and include time (years) as one of the independent variables in the crop-weather model (McKeown et al., 2006). Another important problem in regression models may arise from a high degree of multicollinearity (i.e., significant correlation among one or more independent variables in multiple regression models) (Gommes, 1999), which usually exists between weather variables because monthly temperatures and precipitations are supposed to be highly correlated (Qian et al., 2009; Tannura et al., 2008). When multicollinearity occurs, the estimated regression coefficients are unstable and imprecise which means that the regression model is not reliable. VIF (variance inflation factor) is a wellknown measure of the intensity of multicollinearity and as a rough estimate Gujarati (2003) suggested that multicollinearity is crucial when the correlation coefficient (R) between independent variables is higher than 0.80. To overcome the problem the correlated variables should be omitted from the model following the standard statistical method; some statistical packages (e.g., SigmStat) suggest the candidate variable (s) be eliminated.

FIGURE 9.1 Actual (A) and detrended (B) yield of saffron during the selected period during 1983
2005 for Birjand (an important saffronproducing district in southeast Iran). From Hosseini, M., Mollafilabi, A., Nassiri, M., 2008. Spatial and temporal patterns in saffron (Crocus sativus L.) yield of Khorasan province and their relationship with long term weather variation. Iran. J. Field Crop Res. 6, 79
88 (in Persian).

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Dixon et al. (1994) utilized weather and yield observations of corn at a district level, but homogeneity tests indicated that observations could be pooled at a state level; the same was reported by Tannura et al. (2008). However, the cropweather models developed in saffron-producing regions of Iran showed the best performance at district level, which is seemingly due to the high sensitivity of saffron flowering to temperature (see Section 9.6). Salari et al. (2017) used a 20-year (1990
2010) dataset to develop a multiple regression model for prediction of saffron yield at province level using 76 variables (74 weather variables plus years, and districts). However, it should be noted that running a regression model with time series of only 20 years and 76 independent variables is problematic and the results should be interpreted cautiously. Summing up the results obtained from crop-weather models developed for prediction of saffron yield in Iran, it can be concluded that the minimum (or average) temperatures of April, May, and June (spring) all with negative impact are the best predictors of saffron yield over the studied districts. For example, Hosseini et al. (2008) showed that saffron yield in Birjand (one of the main saffron-producing districts of Iran located in the southeast of the country) could be predicted from the minimum temperatures of April and June (Fig. 9.2). Gommes (1999, 2001) suggested that many of the shortcomings of the crop-weather models can be avoided by using value-added variables such as actual crop evapotranspiration, growth degree days, aridity index, and so on instead of the raw meteorological variables. The word value-added is used because these variables have an agrometeorological basis and include agronomical together with soil and weather information and therefore are superior to raw meteorological variables. It appears that the prediction ability of models could be improved by introducing this type of variable into the regression models of saffron. The accuracy of a crop-weather model in addition to statistics such as coefficient of determination (R2) and correlation coefficient (R) between predicted and actual yields could also be evaluated by using statistical measures such as mean bias, root mean square error (RMSE), or normalized RMSE. Details on the methods for evaluation of model accuracy can found in Bellocchi et al. (2010) and Wallach (2006) among others. Finally, regression models have a limited lifespan and will normally expire after a few years, making a cropweather model older than 3
4 years useless (Gommes, 2001). At this point, the coefficients of the regression should be reestimated assuming the weather variables of the old model are still good predictors of crop yield. However, due to climate change and its consequences on monthly temperature and precipitation the initial weather variables should also be reconsidered.

FIGURE 9.2 Multiple regression model for saffron yield data of Birjand with April and June minimum temperatures as independent variables (R2 5 0.66). Drop lines indicating coordinates of each data point are also shown. From Hosseini, M., Mollafilabi, A., Nassiri, M., 2008. Spatial and temporal patterns in saffron (Crocus sativus L.) yield of Khorasan province and their relationship with long term weather variation. Iran. J. Field Crop Res. 6, 79
88 (in Persian).

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143

Artificial neural networks

Artificial neural networks (ANNs) are powerful computing tools originally developed to create a mathematical model of the human brain. The main objective of an ANN is to establish a system that performs various calculations more accurately and faster than traditional systems (Haykin, 1999). Since 1960, ANNs have been extensively used in different sciences. In agricultural research, ANNs are widely applied for yield prediction from weather variables as an alternative to conventional regression models (Paswan and Ara Begum, 2013). Some researchers have provided detailed comparisons between ANNs and regression models (e.g., Caselli et al., 2009; Cheng and Titterington, 1994; Ripley, 1994; Smith and Mason, 1997); most of them are reviewed by Paswan and Ara Begum (2013). In the following section, some of the results of their application for prediction of saffron yield are presented after a brief review of ANNs, details could be found in Bose and Liang (1996), Cheng and Titterington (1994), and Haykin (1999).

9.3.1 Artificial neural networks, an overview An ANN is basically an architecture representing the structure of a network with its nodes (neurons) and connection lines. These architectures can be categorized into two main groups, feedforward networks and feedback networks, with the first type being the most common in crop yield prediction (Paswan and Ara Begum, 2013). Feedforward networks consist of nodes (processing units) arranged in layers so that nodes of any layer are connected to nodes of the previous layer and the signals can only flow in one direction, from input to output. The strength of node connections is known as weight (w), with higher weights indicating stronger connections. There are two types of feedforward ANNs are divided: 1. A single-layer feedforward network. also known as a single-layer perceptron (SLP), consisting of only one input layer, which is fully connected to the output layer (Fig. 9.3A). 2. A multilayer feedforward network, also known as a multilayer perceptron (MLP), which has one or more layers between the input and the output layer called hidden layer(s) (Fig. 9.3B). The number of nodes in a hidden layer can be less than the number of inputs or outputs. The data processing scheme of a SLP is given in Fig. 9.3C. The inputs (xi) are introduced to the input layer. The input layer is analogous to independent variables in regression models and for crop yield prediction are weather variables. Each node of the input layer has a weight (wi) that shows how strong the node is connected to the other nodes. Weights in ANNs are the same as estimated coefficients in regression models. The net input then is calculated as the sum of the products of each input by its corresponding weight. For each layer of the network a bias node is also required. The bias node in a neural network is a node that is always “on.” That is, its value is always set to one. It is analogous to the intercept in a regression model, and has the same task. Net input is subjected to further processing through an activation function, which can be defined as the extra analytical force applied over the input to obtain an exact output. Different types of activation functions can be used, but the sigmoid (S-shaped) function is the most common type. This activation function generates input values between zero and one (for the binary sigmoid function) or between 21 and 11 (for the bipolar sigmoid function). All forms of sigmoid functions are bounded (asymptotic); that is, the generated output cannot be less than zero or more than one. Finally the output can be calculated by applying the activation function over the net input (Y 5 f(Inet)). The output layer is equivalent to predicted values in regression models and for crop yield prediction is the same as the estimated yield. The output of an ANN (e.g., estimated yield) may be different from the desired output (e.g., actual yield); if so, the weights (coefficients) should be readjusted. Learning (also called training) in ANNs is the method of modifying the weight of connections between nodes in order to reduce the error. Among the different types of learning, supervised learning is the most popular method in crop yield prediction. In this method, the output of the network is compared with the desired output and if there is a difference between estimated and actual output an error signal is generated. On the basis of this error signal, the weights are adjusted and the iteration process is continued until the ANN output matches the actual (or desired) output. Learning of ANNs is analogous to least squares regression. As can be seen, a two-layer, feedforward network with one hidden layer (two-layer MLP), one sigmoid function in the hidden layer, and one output node where weights are adjusted using supervised learning is the best setup for an ANN when crop yield prediction is the objective (Guo and Xue, 2012; Mathieu and Aires, 2016). Evaluation of ANNs is usually conducted in two separate steps: assessment of the learning process and validation of the trained network using independent data points. In practice, all existing data should be divided into two independent (orthogonal) sets. One set will be used for training of the ANN (learning process) and the other for

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FIGURE 9.3 Schematic presentation of feedforward ANNs: (A) SLP with several outputs, (B) the same as A but with one output, (C) MLP with several outputs, and (D) the same as C but with one output. A simple scheme of a learning algorithm for SLPs with one output is also shown in below panel: inputs (xi) will be presented to the network, multiplied by weights (wi), and summed plus a bias term (b), summation goes to a sigmoid activation function (f) to generate the output of the network (Y). During the learning process, Y will be compared with a desired output (Yd) and an error signal will be generated; based on the error, weights will be adjusted. The process is continued until the perfect match between Y and Yd is achieved.

validation/verification: testing of the ANN performance and estimation of the error rate. The performance of an ANN could be tested by the same statistical measures used for evaluation of regression models (e.g., R2, RMSE, etc.).

9.3.2 Application of artificial neural networks for saffron The first attempt to apply an ANN for saffron yield prediction Akbarpour et al. (2013) using an MLP network with monthly temperature, precipitation, and humidity as input variables showed that saffron yield was precisely estimated (R2 5 0.91; RMSE 5 0.2 kg ha21). The authors indicted that precipitation was the most important yield-determining factor followed by temperature and humidity. However, their study was conducted at district level and therefore extending the results to larger scales seems doubtful. Nekouei et al. (2014) applied an ANN for saffron yield prediction at province scale. They developed a multilayer feedforward perceptron with one hidden layer. Using different combinations of monthly minimum and maximum temperatures, precipitation, evapotranspiration, and relative humidity of a 20-year period over two adjacent provinces, they made 86 sets of variables, which could potentially be used as ANN input. Selection of the proper number of inputs with the highest prediction ability over a network is an important task. The Gamma test is a new statistical method for selecting the best set of input variables for nonlinear modeling, and is able to provide the best mean square error that can possibly be achieved using any nonlinear smooth model. In fact, it allows to efficiently calculate that part of the variance

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of the output (e.g., yield) that cannot be accounted for by any model based on the inputs (e.g., weather variables), even though this model is unknown (Jones, 2004). Applying the Gamma test, Nekouei et al. (2014) selected eight sets of weather variables as inputs of an MLP. After training the MLP, the prediction ability of eight networks was evaluated using inputs independent from those used during training. The results indicated that saffron yield in the two provinces was reasonably predicted by all networks. However, the best results (R2 5 0.88; RMSE 5 0.68 kg ha21) were obtained with a set of six weather variables including maximum temperatures of September, October, and November, sum of autumn precipitation, sum of autumn evapotranspiration, sum of autumn relative humidity, and saffron yield of the preceding year. Riahi Modavar et al. (2017) used an MLP with two hidden layers and a sigmoid activation function for prediction of saffron yield at province scale. They first calculated 37 potential input variables based on monthly and seasonal values of minimum and maximum temperatures, precipitation, evapotranspiration, and relative humidity. The candidate variables were then subjected to correlation analysis for selection of the best inputs with the highest correlation with saffron yield and lack of multicolinearity. On this basis, 10 variables were selected as the best subset for running the ANN. The results showed that the MLP was able to precisely predict saffron yield (Fig. 9.4) both in training (R2 5 0.97) and validation (R2 5 0.69). However, the accuracy of prediction was reduced when the performance of the ANN was tested with independent data (Fig. 9.4A and B). This could be due to the method of selection of inputs as well as the number of inputs. While correlation analysis is the conventional method for selection of ANN inputs, it should be noted that Pearson correlation is a measure of linear relationship provided that it cannot detect nonlinear relations between variables, if any (Crawley, 2011). However, the Gamma test covers both linear and nonlinear relationships and seemingly works much better than simple correlation for selection of ANN inputs. On the other hand, the number of input variables is an important issue. Mathieu and Aires (2016) argued that six is the optimal number of inputs for MLPs with one hidden layer. A large number of inputs may lead to overfitting, which means learning insignificant details. Overfit ANNs usually have perfect results in the training phase but show poor performance during validation and verification.

FIGURE 9.4 (A) Comparison between observed and predicted yield of saffron during the training process of ANN. (B) The same comparison as in (A) but during validation of ANN results with independent data. The predication error (difference between observed and predicted yield) is also shown in both (A) and (B). From Riahi Modavar, H., Khashei-Siuki, A., Seifi, A., 2017. Accuracy and uncertainty analysis of artificial neural network in predicting saffron yield in the south Khorasan province based on meteorological data. Saffron Agron. Technol. 5, 255
271 (in Persian).

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Nevertheless, the results reported by Riahi Modavar et al. (2017) are good enough to support the ability of ANNs to reasonably predict saffron yield at regional scale. The higher prediction ability of ANNs over multiple regression models has been confirmed in several studies conducted under different weather conditions and with different crop species. However, ANNs and regression models are competing methods and thus it seems necessary in ANN-based yield prediction research to also run a regression model with the same input variables used in the network. Comparison of the results shows which method outperforms the other under different climatic conditions and spatial scales.

9.4

Application of response surface modeling in saffron production

9.4.1 Statistical background Response surface methodology (RSM) was developed by Box and Wilson (1951) to improve production processes in the chemical industries. The main objective was to optimize chemical reactions to achieve high yield and purity at low cost. This was realized by conducting series of experiments involving factors such as temperature, pressure, duration of reaction, and proportion of reactants. The same methodology can be used to model or optimize any response that is affected by the levels of one or more quantitative factors. The statistical basis of this method is polynomial regression modeling. In this section a brief overview of response surface modeling will be presented; details on the methodology and underlying statistical procedure can be found in several textbooks such as Dean and Voss (1999) and Montgomery (2001). The general setting of the method is as follows: The response is a quantitative continuous variable (e.g., yield, purity, efficiency), and the mean response is an unknown function of the levels of K factors (e.g., temperature, fertilizer), where the levels of these factors are real values and precisely controllable. The factors are arranged in a special type of experimental design that allows for fitting second-order polynomials to the measured response. The mean response, when plotted as a function of the treatment combinations, is a surface called the response surface as shown for two factors (X1 and X2) in Fig. 9.5. Response surface methods may be employed to find factor settings that produce the desired (maximum, minimum, or optimum) response, to find factor settings that satisfy management specifications, and to model the relationship between the quantitative factors and the response.

9.4.2 Central composite designs Central composite designs (CCDs) were first described by Box and Wilson (1951), and practically are the most common response surface designs. CCDs as shown in Fig. 9.6 consist of: 1. 2K factorial points (also called cube points), where K is the number of factors each in two levels, and the factorial points are 4 and 8 for K 5 2 and 3, respectively.

FIGURE 9.5 Schematic presentation of response surface for two factors (X1 and X2) using hypothetical values.

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FIGURE 9.6 CCD with two (A) and three (B) factors. Closed circles are factorial points, open circles are axial points, and the closed circle in the middle labeled with C is the center point.

2. Axial points (also called star points) are points located at a given distance α from the design center in each direction on each axis to allow estimation of curvature. Thus if there are K factors, there are 2K axial points. While the choice of α depends on the type of design, a common value is α 5 (2K)1/4. Axial points are 4 and 6 with α 5 1.414 and 1.682 for K 5 2 and 3 designs, respectively. The axial points are usually outside the cube (Fig. 9.6), but they could be inside the cube (for α , 1) or on the cube (for α 5 1). A special type of CCD with α 5 1 is the face-centered design where the axial points are at the center of each face of the factorial space. 3. Center point designs should include enough replication, often at the center points, to allow checking the accuracy of the model (lack of fit). The default number of center points is 5 and 6 for K 5 2 and 3 designs, respectively. The sum of factorial points, axial points, and center points determines the total number of experimental runs that should be performed, which are 13 (4 1 4 1 5) and 20 (8 1 6 1 6) for K 5 2 and 3 designs, respectively. In a response surface design, for each factor a low level and a high level should be defined; the low level of all factors is coded to 21 and the high level to 11, then the center point will be the average of the two levels and is coded to 0. For instance, in a CCD with two factors (nitrogen: 0 and 100 kg ha21; density: 5 and 15 plants m22), the factor combination coded as (21, 11) corresponds to 0 kg ha21 nitrogen and 15 plants m22. The center point coded as zero is 50 kg ha21 nitrogen and density of 10 plant m22, then the axial points are at distance 6 α from the design center. For data analysis, a second-order model should be fitted. For K factors, the standard second-order polynomial model is: Yx;t 5 b0 1

K X i51

bi x i 1

K X i51

bii xi 2 1

X

bij xi xj 1 εx;t

(9.1)

i,j

where Yx,t is the tth response observed for the combination of factors (X1, X2, . . ., XK). The random-error variable εx,t is assumed to be independent with N (0, σ2) distribution. The parameter bi represents the linear effect of the ith factor, the parameter bii is the quadratic effect of the ith factor, and bij denotes the cross-product effect, or interaction effect, between the ith and jth factors. The polynomial model (Eq. 9.1) is subjected to analysis of variance to test the significance of the linear, quadratic, and interaction terms. Validation of the final model including all significant terms against the observed response should be conducted using statistical measures (e.g., coefficient of determination (R2) or RMSE). The validated regression model is a powerful tool for setting factor levels to achieve the desired response depending on the defined specification. Since each response is important for determining the desirability of a system, we need to consider all responses simultaneously. For example, to increase the yield and decrease the environmental impacts of a production system, optimal settings of the factors for one response may be far from that of another response. Response optimization is a method that allows for tradeoffs among the various responses and identifying the combination of factor settings that jointly optimize a set of response variables. Response surface designs can be readily analyzed using statistical packages such as SAS, MINITAB, STATISTICA, or Design Expert.

9.4.3 Application While RSM is widely applied to industrial studies, its application in agricultural sciences is rather new. Weed competition in rice fields was studied by Pantone and Baker (1991) using response surface analysis. Dhungana et al. (2006)

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TABLE 9.1 Actual and coded values of two factors (X1 and X2) arranged in a central composite design. Runs

Corm weight (g corm21) X1

Corm density (corm m22) X2

Corm weight (coded) X1

Corm density (coded) X2

1 2 3 4 5 6 7 8 9 10 11 12 13

3 7 3 7 3 7 5 5 5 5 5 5 5

50 50 250 250 150 150 50 250 150 150 150 150 150

21 11 21 11 21 11 0 0 0 0 0 0 0

21 21 11 11 0 0 21 11 0 0 0 0 0

Runs 1
4 are factorial points, 4
8 axial points, and 9
12 center points in 5 replications; it should be noted that the design is face centered with α 5 1. Source: From Nassiri Mahallati, M., Koocheki, A., Amin Ghafouri, A., Mahluji Rad, M., 2015. Optimizing corm size and density in saffron (Crocus sativus L.) cultivation by central composite design. Saffron Agron. Technol. 3, 161
177 (in Persian).

applied a simulation together with response surface models to design wheat production systems for future climates. The same authors (Dhungana et al., 2008) used response surface analysis to optimize wheat response to climate change. RSM was also used by Koocheki et al. (2014) for optimization of canola yield response to water, nitrogen, and plant density under different production scenarios and by Muriithi (2015) to optimize yield response of potato tuber yield. Nassiri Mahallati et al. (2015) applied RSM to analyze the yield of saffron in response to corm size and corm density. They used a two-factor CCD where the higher and the lower levels of corm size and corm density was set as 3
7 g corm21 and 50
250 corm m22, respectively. The design with five replications for center points included 13 runs as shown in Table 9.1. A field experiment with 13 combinations of two factors (Table 9.1) was conducted and several response variables (e.g., economic yield of saffron (dry stigma yield), number of daughter corms, corm diameter) were measured. Secondorder polynomial regression plus interaction effect (Eq. 9.1) was fitted to the measured data and the significance of linear, quadratic, and interaction terms for all responses were tested by performing analysis of variance. The response surface plots of saffron economic yield and density of daughter corms obtained by fitting the full quadratic model are shown in Fig. 9.7A and B, respectively. The results indicate that both responses were precisely predicted by the quadratic model (R2 5 0.96 and 0.93 for saffron yield and corm density, respectively) and therefore could be used for further analysis. The contour plot (projection of surface on a 2D plane) of saffron yield (Fig. 9.7C) could be used to understand the interaction effect of factors on response variables by following the desired isoline (contour line with the same response value). For instance, in Fig. 9.7C a saffron yield of 6000 g ha21 will be obtained by planting corms with 12 g weight at the density of 120 corm m22. However, the same yield could be achieved from smaller-sized corms (e.g., 7 g) when sown at higher density (e.g., 200 corm m22). Optimizing models have the specific objective of identifying the best option in terms of management inputs for practical operation of a production system. However, optimizing models do not allow the inclusion of many biological details and may be poor representations of reality. Using the crop simulation models to identify a limited set of management options that are then evaluated with the optimizing models has been reported as a useful approach to overcome this shortcoming (Dhungana et al., 2006).

9.5

Dynamic simulation models

Multiple regression models and ANNs, as discussed in the previous sections, are able to precisely predict crop yield based on several weather variables. However, they just provide mathematical relations between input and output variables, telling us nothing about the underlying mechanisms. Mechanistic simulation models are more sophisticated tools for prediction of crop yield based on growth processes such as leaf area expansion, dry matter production, and

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FIGURE 9.7 Response surface plots for economic yield of saffron (A) and density of daughter corms (B) generated by fitting the full quadratic model; the contour plot of saffron yield is also shown (C). From Nassiri Mahallati, M., Koocheki, A., Amin Ghafouri, A., Mahluji Rad, M., 2015. Optimizing corm size and density in saffron (Crocus sativus L.) cultivation by central composite design. Saffron Agron. Technol. 3, 161
177 (in Persian).

phonological development and interaction of these processes with environmental factors (Van Ittersum et al., 2003). Such models are able to simulate the time course of crop growth and development in daily intervals and therefore are known as dynamic simulation models. Environmental factors that affect the growth and developmental processes of plants are solar radiation, temperature, water, and nutrients and quantifying their effects on plants is the core of mathematical models of crop production (Janssen et al., 2017). Growth (i.e., dry matter production) is the main physiological process in any simulation model, and is usually calculated based on the absorbed resource using resource use efficiency, which converts the amount of absorbed resource into the dry matter. Solar radiation and water are the most important resources used as the basis of dry matter production in different simulation models. In radiation-based models, a detailed description of canopy structure is required for calculation of absorbed radiation. However, when water is considered as the main resource for dry matter production, belowground processes (e.g., root growth and soil water balance) as well as crop transpiration should be determined precisely (Janssen et al., 2017; Ritchie and Alagarswamy, 2002). The mathematical structure of simulation models is rather complicated and a large number of calculations should be performed in daily intervals over the whole growth period (emergence to harvest); however, programming languages

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such as Fortran or C11 with powerful mathematical libraries provide the required facilities for running these simulation models. Daily weather data (e.g., temperature, precipitation, radiation), several soil- and crop-specific parameters, and management information (e.g., sowing date, planting density, and harvest date) are the main inputs of simulation models. The outputs of crop models are highly dependent on the quality of inputs and thus setting the correct values for model parameters is an important job in modeling studies. Theses parameters are usually obtained by direct measurements or may be collected from the literature. Before application, crop modes should be passed through two separate stages: calibration and validation. During calibration model parameters are adjusted to obtain perfect accordance between model outputs (e.g., dry matter or grain yield) and the measured values of the same outputs. The calibrated model should also be validated for its accuracy with fixed parameters and a set of independent data from the dataset used in calibration. In both calibration and validation the difference between measured and simulated outputs is compared by using statistical measures such as R-squared, RMSE, normalized RMSE (NRMSE). Details on statistical methods for validation of crop models can be found in Bellocchi et al. (2010) and Wallach (2006). Since 1965, a large number of crop models have been developed for simulation of growth, development, and yield of different crop species. Jones et al. (2017) reviewed the history of crop simulation models and found that most modeling studies focus on a few major crop species. However, the modeling techniques are applicable for any plant species including locally important crops such as saffron.

9.5.1 Advances in development of simulation models for saffron As the first attempt toward modeling saffron growth, Sepaskhah et al. (2013) developed a dynamic water-based model for prediction of saffron yield (corm and stigma) and other growth variables. The model has an interactive graphic interface and requires readily available input data. Daily values of minimum and maximum temperature, minimum and maximum relative humidity, precipitation, wind speed, and sunny hours should be prepared in a predefined format as an input file. Constant parameters such as geographical coordinates, soil water content at field capacity, and permanent wilting point (both are functions of soil texture) can be easily changed by the user. The model starts with calculation of daily values of reference evapotranspiration (ETo, mm d21) using three standard methods: FAO
Penman, Penman
Monteith, and Hargreaves
Samani. ETo is then multiplied by the crop coefficient (Kc) resulting in the daily crop evapotranspiration (ETc, mm d21). Specific Kc values for saffron are estimated from an empirically derived polynomial regression model (Eq. 9.2) between Kc (estimated as the ETc/ET ratio) and the number of days after first irrigation (DAFI): Kc 5 a0 1 a1 ðDAFIÞ2 1 a2 ðDAFIÞ3 1 a3 ðDAFIÞ4

(9.2)

The coefficients of the regression model (a0
a3) are different for the first 3 years after sowing but remain constant afterward. Following the method described by Allen et al. (1998) daily rates of actual crop evapotranspiration (ETa, mm d21) were estimated from ETc using a water stress factor (0 # Ks $ 1), which accounts for water deficits during the growth season. Ks was estimated based on detailed calculations of four-layer soil water balance at the rooting depth of saffron. Seasonal soil evaporation (E, mm) was then subtracted from ETa to obtain seasonal transpiration (T, mm) where E is calculated as a function of leaf area index (LAI). Daily values of LAI were obtained by two different regression equations assuming that until 150 DAFI LAI was a function of daily ETa and afterward decreased with DAFI. Finally saffron corm yield (B, kg ha21) was calculated on the basis of seasonal transpiration (B 5 57.18 3 T) where 57.18 is an empirically derived transpiration efficiency (kg ha21 mm21). Saffron (stigma) yield (Y, kg ha21) was then obtained empirically as a function of corm yield (Y 5 4.31 3 1022 3 B2). It should be noted that the coefficients of regression equations defined in the model were estimated using data collected from previously conducted experiments on saffron under water-stressed and nonstressed conditions. Therefore the model is capable of covering a wide range of climatic conditions. The model generates an output file with six tables (e.g., ETo table, LAI table, yield table). The model was perfectly calibrated for prediction of the components of soil water balance, LAI, corm, and stigma yield of saffron using data from three different experiments conducted in a semiarid region. The calibrated model was then validated against data from two independent experiments conducted in a semiarid and an arid region. The performance of the calibrated model for prediction of saffron yield is shown in Fig. 9.8. Stigma yield was reasonably simulated both in arid and semiarid regions (R2 5 0.98, NRMSE 5 13%) showing the high prediction ability of the model over a wide range of climatic conditions.

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FIGURE 9.8 Relationship between measured (circle, arid region; triangle, semiarid region) and predicted saffron yield in the third growing season. From Sepaskhah, A.R., Amini-Nejad, M., Kamgar-Haghighi, A.A., 2013. Developing a dynamic yield and growth model for saffron under different irrigation regimes. Int. J. Plant Prod. 7, 473
504.

FIGURE 9.9 Measured values and simulated time course of LAI under different irrigation regimes (100%
50% of ETc) as well as in rainfed conditions (no irrigation) for the third growing season of saffron in a semiarid region. From Sepaskhah, A.R., Amini-Nejad, M., Kamgar-Haghighi, A.A., 2013. Developing a dynamic yield and growth model for saffron under different irrigation regimes. Int. J. Plant Prod. 7, 473
504.

The simulated LAI of saffron was also perfectly validated against measured values under different irrigation regimes and under rainfed conditions (Fig. 9.9) with overall R-squared of 0.94 between observed and simulated values and RMSE of 0.32. AquaCrop is a crop water-productivity model developed by FAO in 2009 that has been widely used for research and management purposes for different crops and under different climatic conditions. AquaCrop is a free and welldocumented model; details on the structure and calculation methods of the model can be found in Raes et al. (2009, 2017) and Steduto et al. (2009). In brief, AquaCrop is based on the Doorenbos and Kasssam (1979) model (Eq. 9.3), which describes crop yield response to water stress:     Y ET 12 5 Ky 1 2 (9.3) Ym ETm

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where Ym and Y represent the maximum and actual yields, ETm and ET the maximum and actual evapotranspiration, and Ky is a factor that relates relative yield reduction to the relative reduction in ET. In AquaCrop, two important changes are made to Eq. (9.3): the nonproductive component of evapotranspiration (i.e., evaporation) is subtracted from ET and yield is defined as the product of biomass (B) by harvest index (HI) and then B is calculated from Eq. (9.4): X B 5 WP 3 Tr (9.4) where B is biomass (kg m22), Tr (ET minus evaporation) is daily transpiration (mm), and WP is water productivity (kg biomass m22 mm21 cumulative Tr over growth season). WP is further adjusted for the evaporative power of the atmosphere, CO2 concentration, soil fertility, and chemical composition of biomass. With normalized (adjusted) WP or WP* biomass is defined by Eq. (9.5): X  Tr  B 5 WP  (9.5) ETo where the reference evapotranspiration (ETo) is introduced to adjust for evaporative power. It should be noted that the unit of normalized WP* is kg m22 or kg ha21. Details on normalization methods of WP are described by Raes et al. (2017). The value of WP* for C4 species is in the range of 30
35 and for C3 species 15
20 g m22. AquaCrop uses a default value of WP* and HI for different annual crops, but these values are not available for perennial species including saffron. Using experimental data collected over six successive years of saffron growth Mirsafi et al. (2016) adapted AquaCrop for simulation of saffron yield and dry matter production in a semiarid region of Iran. They estimated separate values of WP* and HI for each growth year of saffron assuming that saffron yield in each given year is a function of corm yield in the previous year. However, the estimated values of WP* (3.7 g m22) and HI (0.35%) were extremely different from the values reported for annual crops. AquaCrop was then calibrated and validated against independent data. The results indicted the reasonable performance of the calibrated model for simulation of soil water content (NRMSE 5 14%), saffron yield (NRMSE 5 11% pooled over 6 years), and biomass (overall NRMSE 5 25%). The authors stated that the model showed high precision in the first years of saffron growth, but accuracy declined with increasing the age of the saffron field. Both AquaCrop and the model of Sepaskhah et al. (2013) are water-based models where dry matter production is simulated based on crop water productivity. In fact, the transpiration efficiency (58.17 kg m22 mm21) used by Sepaskhah et al. (2013) for calculation of saffron biomass is the same as the WP in AquaCrop before being normalized. However, growth (dry matter production) could also be estimated based on the amount of absorbed radiation by the crop canopy. In the following section the structure of a simple radiation-based model is presented for dynamic simulation of dry matter production of saffron.

9.5.2 Radiation-based model for saffron growth While temperature is the main driver of developmental processes, the total amount of absorbed radiation is the key determinant of plant growth and dry matter production. On this basis the first step in simulation of dry matter production of a crop species is prediction of the daily amount of absorbed radiation by the crop canopy. In the next step, absorbed radiation is converted to dry matter using two basic methods. In the first method radiation energy is saved as chemical energy (i.e., gross assimilation) through photosynthetic machinery. Part of the gross assimilation is used in crop maintenance processes and the remaining is converted to dry matter through growth respiration. This method is used in some well-known crop simulation models such as SUCROS (Van Laar et al., 1997), WOFOST (Boogaard et al., 1998), and ORYZA (Bouman et al., 2001); the mathematical structure of these models is given by Van Ittersum et al. (2003). However, in this approach detailed information and data about assimilation rate, maintenance, and growth respiration of crop species is required, which is not easily available for local species such as saffron. In the second method, absorbed radiation is directly converted into dry matter by using a measure of net assimilation known as radiation use efficiency (RUE). Radiation use efficiency, first defined by Monteith (1977) as a robust cropspecific parameter, led to the development of simple and at the same time accurate models (Sinclair and Muchow, 1999; Sto¨ckle and Kemanian, 2009). This approach for simulation of dry matter production is widely used in many crop models such as CERES (Ritchie and Alagarswamy, 2002), APSIM (Keating et al., 2003), and LINTUL (Van Delden, 2001) and would be an appropriate method for modeling growth processes of local crop species.

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9.5.2.1 Structure of a radiation-based model Crop growth refers to the accumulation of dry matter (DM, g m22), also known as biomass, and crop growth rate is defined as DM production per day (g m22 d21). DM refers to aboveground parts of the crop because the dry weight of roots is difficult to measure; however, for crop species with belowground economic parts (e.g., potato, sugar beet) the dry weight of storage organs is included in DM. In RUE-based models it is assumed that the crop growth rate under optimal conditions is proportional to the amount of solar radiation intercepted by the canopy (Monteith, 1977). On this basis dry matter production is simulated as the product of intercepted radiation and RUE. The produced dry matter is partitioned to shoots (i.e., leaves and stems) and storage organs (e.g., corms in saffron) using partitioning factors defined as a function of the development stage of the crop. The accumulated DM of plant organs is calculated by integration of their growth rates over time with daily intervals.

9.5.2.2 Leaf growth and senescence The area of green leaves within the crop canopy is the main determinant of light interception and utilization. The crop canopy is usually quantified using the LAI (m2leaf m22ground). In the model, LAI is simulated by integrating the growth rate of the LAI (m2 m22 d21), which is the difference between the growth rate of green LAI (LAIG, m2 m22 d21) and the rate of leaf senescence (LAID, m2 m22 d21): Ð dLAI 3 dt 1 LAI0 dt dLAI dLAIG dLAID 5 2 dt dt dt

LAI 5

(9.6)

The growth of green leaf area is calculated by multiplying the simulated crop growth rate (gDM m22 d21) by the leaf weight ratio (LWR, gleaf g21DM) and by the specific leaf area (SLA, m2leaf g21leaf) (Eq. 9.7): dLAIG dDM 3 LWR 3 SLA 5 dt dt

(9.7)

The senescence rate of the LAI is described as the product of the relative death rate of leaves (RDR, day21) and LAI: dLAID 5 LAI 3 RDR dt

(9.8)

In the simulation models, the RDR of leaves is either due to aging or due to self-shading. Self-shading usually occurs in dense canopies (LAI . 4) and therefore is unlikely in open canopies such as that of saffron. However, aging will result in leaf death after a given stage of crop growth defined by degree days. RDR is a function of the average daily temperature and results in a decrease of LAI toward the end of the growing season after approaching a maximum. It should be noted that RDR is a relative measure; for instance, RDR 5 0.02 day21 means that each day 2% of the existing LAI will be lost.

9.5.2.3 Radiation absorption and dry matter production Light usually refers to the visible part of the total solar radiation; this part (wavelengths between 400 and 700 nm) is also the spectrum that activates the plant photosynthetic system and therefore is called photosynthetically active radiation (PAR). The fraction of PAR is about 50% of the daily total radiation, which is almost constant over any atmospheric conditions. The daily values of absorbed photosynthetically active radiation (PARa, MJ m22 d21) is an exponential function of the LAI: PARa 5 0:5 3 I0 3 ð1 2 eð2K 3 LAIÞ Þ 22

21

(9.9)

where I0 is the daily global radiation (MJ m d ) and K is a crop-specific light extinction coefficient showing the fraction of PAR intercepted per unit LAI. The daily growth rate of the crop (g m22 d21) is then calculated by multiplying the daily amount of absorbed PAR by the RUE (g MJ21) (Eq. 9.10) and DM (g m22) is obtained by integration of growth rate over time (Eq. 9.11):

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dDM 5 PARa 3 RUE dt ð dDM Udt 1 DM0 DM 5 dt

(9.10) (9.11)

9.5.2.4 Inputs and parameters of the model The RUE-based model described in Section 9.5.2 is structured based on the LINTUL model (Van Delden, 2001). Weather inputs are daily minimum and maximum temperatures and radiation; for water-limited conditions daily precipitation and relative humidity are also required. Daily radiation could be calculated from incoming solar radiation adjusted for sunny hours (which is routinely measured in weather stations) using any standard software (e.g., RadEst; Donatelli et al., 2003). Crop-specific parameters of saffron could be collected from published literature, but so far limited data are available. Mirhashemi (2015) reported some of these parameters measured during a 2-year field experiment. Light extinction coefficient (K) and RUE are two model parameters but their values are not available for saffron. Mirhashemi et al. (2015) reported an average K of 0.6 for saffron and discussed that the coefficient is higher in the first growth season. However, the RUE of saffron was lower in the first growth season with average values of 0.68 and 1.73 g MJ21 PAR for the first and the second years of growth, respectively (Mirhashemi et al., 2015).

9.6

Modeling saffron development and flowering

9.6.1 Hypothetical model of saffron development From the modeling point of view for understanding the whole crop system submodels for development (flower initiation and flower emergence) and vegetative growth of saffron are required. However, in the water-productivity models described in the previous section developmental processes were not included. Since flowering behavior plays an important role in saffron yield formation, in this section a model for simulation of saffron flowering is presented. Before developing a mathematical model, it is helpful to create a simple presentation of the main processes that should be included in the model. To create such a hypothetical model the main subprocesses during the annual lifecycle of saffron at field level are considered. It should be noted that at this point the physiological basis of these processes is not required. The lifecycle of saffron consists of two quite different parts that occur above- and belowground. The aboveground processes start by flower emergence (the unique feature of saffron among cultivated crops) in autumn followed by vegetative growth in winter, and ends in spring with the formation of new corms, so-called daughter corms (Fig. 9.10). The belowground phase starts with newly formed dormant corms followed by flower initiation processes (invisible at field) during the high temperatures of summer (Molina et al., 2004). The length of lifecycle events as shown in Fig. 9.10 has high variation depending on environmental conditions and temperature changes. For example, in the growing areas of Khorasan province, the major saffron-producing region of Iran, flowers usually appear during October to November (Kafi et al., 2005). Because flower emergence in saffron is highly dependent on temperature, a thermal gradient from the north to the south of the country has caused a 1-month extension of the flowering period over the province. In other words, flowers first appear in the northern regions where FIGURE 9.10 Hypothetical model of development (flower initiation and flower emergence) and vegetative growth of saffron; timing of the events is also shown. f1 and f2 are temperature functions for flower initiation and emergence, respectively (see text for details).

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low temperatures required for flower emergence occur earlier in autumn and then in the southern parts where the optimum temperature for flower emergence occurs up to 30 days later (Kafi et al., 2005; Koul and Farooq, 1984). However, flower initiation starts much earlier and depending on the regional temperature lasts from early spring until midsummer in different saffron-producing countries (Azizbekova and Milyaeva, 1999; Farooq and Koul, 1983; Juana et al., 2009) and from May to mid-June in Iran (Behdani, 2005). Variability of developmental stages over a spatial scale has also been reported for other crops. As an example Porter et al. (1987) noted that for a given variety of winter wheat (Triticum aestivum) time to grain initiation, flowering, and the end of the grain filling period differed by 30, 20, and 30 days, respectively, in 10 sampling sites from the north to the south of the United Kingdom. The same is true for a given region where year-to-year variation of temperature may affect the timing of developmental events.

9.6.2 Developmental responses in crop species Development is defined as successive morphological events during the lifecycle of plants. On this basis developmental stages are distinguished either through changes in the number (not the size) of plant organs such as number of leaves, or through the time taken for a given morphological event such as flowering (Yan and Wallace, 1998). Among environmental factors, temperature is considered as the main driving force of development processes. Progress toward a developmental event (e.g., flowering) is usually accelerated by a temperature increase between a base value (Tb), at which no development occurs, and an optimum (To). It should be noted that responses to this suboptimal range of temperature may be modified by day length in photoperiod-sensitive species. Beyond the optimum temperature extra warming causes decelerated progress in development up to a ceiling temperature (Tc) where the process ceases (Yan and Hunt, 1999). All mathematical models of plant development could be summarized by the base, the optimum, and the ceiling temperatures known as cardinal temperatures. Using these concepts and the hypothetical model of Fig. 9.10, a simple mathematical model for flower initiation and emergence in saffron was developed by Koocheki et al. (2008) and will be presented in the following section with some modifications.

9.6.3 Structure of the model The mode simulates flowering stages of saffron from zero to two, zero being termination of the dormancy period, one being the completion of the first phase of flowering (i.e., flower initiation), and two being the stage of flower emergence. For instance, the development stage 5 0.6 means that flower initiation already started and 60% of the period has passed; similarly stage 5 1.8 means that 80% of the flower emergence period has passed. The length of the development period (d) is the number of days between two consecutive developmental stages and the inverse of d is the development rate: DR 5

1 d

(9.12)

where DR is the development rate (day21), which is somewhat difficult to understand and requires more explanation. Suppose that at a constant temperature period between the end of flower initiation (development stage 5 1 as described above) and flower emergence (development stage 5 2) of saffron is measured as 50 days (d 5 50). Then the development rate (DR) is 1/50 5 0.02 day21, which means that progress in development from stage 1 toward stage 2 is 0.02 per day (i.e., 2% per day). However, temperature is not constant at field level and thus DR should be calculated each day as a function of daily average temperature. In the model the development stage is calculated by the integration of the development rate over time with daily intervals. For better understanding of the model structure, mathematical aspects are presented in two separate sections.

9.6.3.1 Flower initiation After a dormancy period, the first phase of flowering (flower initiation) starts from late spring and requires high temperature (Fig. 9.10). This phase consists of 10 subphases, which can only be identified by microscopic investigation and can be differentiated by formation of flower parts at microscopic scale (Benschop, 1993). Therefore experimental data at field level is not available during this period of development. Under controlled conditions Molina et al. (2004) reported 9 C as the base temperature (where development rate is zero) for flower initiation and 23 C
25 C as the optimum temperature during this period. They showed that under optimal temperature, 100
150 days is needed for

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completion of this phase. Temperature rise above the optimum leads to delayed initiation of flowers and at temperatures higher than 30 C flower initiation ceases (Molina et al., 2004). Development rate is highly temperature dependent and as discussed before the maximum development rate (DRm) will be reached at an optimum temperature (To). Any change in temperature will affect the DR proportional to its maximum. In the model, the effect of temperature on DR during flower initiation period is calculated using a temperature function: DR1 5 DRm1 3 ft1

(9.13)

where ft1 is the temperature function for the flower initiation (Fig. 9.10) and DRm1 is the maximum DR at optimum temperature. Dividing the DR by the maximum rate (DRm) will change ft to a relative value between zero and one. ft1 is defined as a linear function of mean daily temperature (Ta,  C) during the period of flower initiation: ft1 5 1 ft1 5 Tb1 1 bTa ft1 5 Tc1 1 cTa ft1 5 0

if if if if

Ta 5 T01 Tb1 , Ta , To1 To1 , Ta , Tc1 Ta $ Tc1

(9.14)

On this basis if daily average temperature equals optimum temperature (To1,  C), the value of the function equals one and therefore development proceeds with the maximum rate (DRm). DR is zero when Ta equals the base temperature (Tb1,  C) and increases linearly with Ta at suboptimal temperatures between Tb1 and To1. If Ta rises above To1, the value of the function becomes less than one and the DR will be reduced linearly in supraoptimal temperatures between To1 and ceiling temperature (Tc1,  C), and eventually at Tc1 the function equals zero. To1 and Tc1 were chosen from the results reported by Molina et al. (2004) in a controlled environment. In Fig. 9.11 changes in ft1 in response to daily average temperature together with cardinal temperatures for flower initiation are shown.

9.6.3.2 Flower emergence The second phase of saffron development starts after the flower initiation period and requires lower temperature for flower emergence. Under controlled conditions the optimum temperature for this period has been reported to be 15 C
17 C, in this range temperature requirements for flower emergence will be met in 40 days (Molina et al., 2005). Similar to flower initiation, the DR during the flower emergence period (DR2) is calculated by: DR2 5 DRm2 3 ft2

(9.15)

where DRm2 is the maximum value of DR2 and ft2 (Fig. 9.10) is the temperature function during the flower emergence period. While measuring the DR for the flower initiation stage was not possible at field level, for the second phase of flowering where the field data on time to flower appearance is available, the DR could be defined as a function of daily average temperature (Ta). Considering its flexibility, the five-parametric beta function (Yin et al., 1995) was used for estimation of DR2:

FIGURE 9.11 Temperature function (ft1) during the flower initiation period. Values of the function are calculated from the ratio of DR at each temperature to the maximum development rate (DRm) at the optimum temperature. Cardinal temperatures (i.e., Tb1, To1, and Tc1) are defined as reported by Molina et al. (2004). From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

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DR2 5 expðμÞ 3 ðTa 2Tb1 Þα 3 ðTc2 2Ta Þβ

157

(9.16)

where Tb2 and Tc2 are the base and the ceiling temperatures for development during flower emergence period, respectively, and μ, α, and β are the coefficients of the beta model. In this way DR2 can be estimated in a temperature range between Tb2 and Tc2 and the temperature function during this period (ft2) can readily defined by: ft2 5 0 ft2 5 exp ðμÞ 3 ðTa 2Tb2 Þα 3 ðTc2 2Ta Þβ ft2 5 0

if if if

Ta # Tb2 Tb2 , Ta , Tc2 Ta $ Tc2

(9.17)

The value of the function at temperatures less than Tb2 and higher than Tc2 is equal to zero and between these two temperatures is calculated by a nonlinear beta function in such a way that at optimum temperature ft2 equals 1 and therefore DR2 and hence the DR approaches the maximum (DRm2, Eq. 9.15). The optimum temperature for flower emergence period (To2) could be also estimated when the first derivative of the beta function equals zero (Eq. 9.18): To2 5

ðα 3 Tc1 Þ 1 ðβ 3 Tb1 Þ α1β

(9.18)

The maximum temperature for development at this phase (DRm2) is reached when Ta 5 To2 and can be obtained by substituting To2 for Ta in Eq. (9.16), which results in:   Tc1 2Tb1 α1β DRm2 5 exp ðμÞ 3 αα 3 β β (9.19) α1β The beta function fitted perfectly (R2 5 0.91) to the data from four major saffron-producing regions of the Khorasan province (Fig. 9.12A) and Tb2, Tc2, and To2 for the flower emergence period were estimated as 15.9 C, 22.4 C, and 17.9 C, respectively (Table 9.2) with a maximum development rate (DR2) of 0.0289 day21. The inverse of the DR gives the duration of the flower emergence period, which is almost 34 days at optimum temperature, and in the range of average temperatures shown in Fig. 9.12A takes from 34 to 48 days (Fig. 9.12B). It should be noted that at Tb2 and Tc2 DR is zero and hence the duration of flowering period is infinity. The beta function is a flexible nonlinear model and in previous studies was successfully used for modeling the development of crop species including rice (Yin et al., 1995) and vegetables (Yan and Hunt, 1999). However, much easier functions such as bilinear (Craufurd et al., 1998) and second- and third-order polynomials (Yan and Wallace, 1998; Stewart et al., 1998) may also be applied to estimate cardinal temperatures. The cardinal temperatures presented in Table 9.2 are to some extent different from those reported by Molina et al. (2005). They reported 15 C
17 C as the optimum and 23 C as the ceiling temperature for the flower emergence period of saffron. In Fig. 9.13 the temperature function for this period (ft2) predicted from field data using the beta function is compared with those reported by Molina et al. (2005) in a controlled environment with constant temperature. The

FIGURE 9.12 (A) Relationship between DR and daily average temperature for the flower emergence period. Cardinal temperatures for this phase were calculated by fitting the beta function to the data from fields located in four saffron production regions. (B) Relationship between duration of development and temperature. Duration was calculated as the reciprocal of DR for each production region. From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

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TABLE 9.2 Predicated values of the coefficients of the beta function. Coefficient

Value

Standard error

μ Tb2 α Tc2 β To2 DRm2

2 6.092 15.49 0.563 22.41 1.355 17.89 0.0289

2.72 1.16 1.10 1.70 1.73 1.62 0.004

Tb2 and Tc2 are base, maximum, and ceiling temperatures during the flower emergence period, To2 is the optimum temperature ( C) for this phase, and DRm2 is the maximum development rate (day21) at optimum temperature, which are calculated, respectively, from the Eqs. (9.8
9.10). Source: From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

FIGURE 9.13 Variation of temperature function (ft2) for the flower emergence period of saffron in response to temperature. Values of the function are calculated by the ratio of development rate at each temperature to the maximum development rate at the optimum temperature. (A) data from Molina, R.V., Valero, M., Navarro, Y., Guardiola, J.L., Garcıa-Luis, A., 2005. Temperature effects on flower formation in saffron (Crocus sativus L.). Sci. Hortic. 103, 361
379 and (B) estimated using beta function. From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

ceiling temperature is similar, but the optimum and the base temperatures estimated in field conditions are respectively 2 C and 4 C higher than those reported in the controlled environment. This discrepancy is probably due to variability of field data as well as genotypic differences in temperature response of flowering between Spanish and Iranian saffron.

9.6.4 Simulation of saffron response to climate change 9.6.4.1 General impacts of global warming Agriculture activities have been considerably affected by climate change with negative impacts on growth and development of food crops and cropping systems both at regional and global scales (IPCC, 2013; Parry et al., 2004). Plants have adapted to the surrounding environment over long historical periods and any sudden change in the environment will impact their growth pattern. Hence, based on the severity of such changes spatial and temporal shifts in the historical production regions may occur and elimination from the adapted cropping system would be inevitable (Horie et al., 2000). Temperature is the most important factor affecting physiological processes such as photosynthesis, respiration, and development of crop species (Atkinson and Porter, 1996). Temperature rise will change the flowering pattern of plants (Chmielewski et al., 2003; Fulu et al., 2006), but little has been done to investigate such an event for locally important crops. During recent years smaller harvests of saffron flowers in the main production areas of Iran have been observed, which could be related to the impact of climate change. While agronomical practices such as selection of corm size,

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age of the stand, planting methods and density, and irrigation interval are reported to be the causes of yield variation, temperature seems to be the most important environmental factor in determining yield due to its effect on the flowering behavior of saffron (Gresta et al., 2009; Molina et al., 2004, 2005). Temperature rise and its impact on field crops has been researched in Iran (Koocheki et al., 2006; Nassiri et al., 2006), but such an investigation is new for saffron. It is assumed that climate change and temperature rise will have a significant direct effect on saffron production in the future and a shift in date of planting and area of production seems to be inevitable. It should be noted that the same variation in the timing of growth events of saffron would be possible in a given region due to year-to-year variability of temperature. Furthermore, this variability is expected to increase by global warming.

9.6.4.2 Effects of climate change on development rate Using the development model presented in Section 9.6.3, Koocheki et al. (2008) simulated the effects of temperature rise on the flowering response of saffron for 0.5 C, 1 C, 1.5 C, and 2 C increase in daily average temperature in four regions of Khorasan province, Iran. With increasing daily average temperature DR at both phases of flowering was reduced considerably (Fig. 9.14). However, the maximum reduction of DR with each 1 C increase in mean daily temperature was to some extent higher for flower emergence compared with flower initiation. Variation of the simulated ft2 function (Fig. 9.15) shows that with increasing mean daily temperature, the function deviated from the optimum value (ft2 5 1) and with temperature rise by 1 C
2 C above the current temperature ft2 reached 0.8 and 0.65, respectively. Therefore increased temperature due to climate change will be led to occurrence of supra optimal temperatures during the flower emergence period of saffron which results to lowering development rate.

FIGURE 9.14 Simulated values of DR for flower initiation (solid circles) and flower emergence (open circles) periods in response to 0.5 C
2 C increase of mean daily temperature above the present conditions. Vertical bars are standard error of mean over studied regions. From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

FIGURE 9.15 Simulated values of temperature function in the second phase of flowering (ft2) with mean daily temperature increase of 0.5 C
2 C above the present values considering the effects of elevated temperature on DR in the first phase (ft1). From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

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Results from different studies indicated that climate change and global warming will increase the length of growing season in different parts of the world (Chmielewski and Ro¨tzer, 2002; Menzel and Fabian, 1999). On the other hand, with increasing temperature in the range between base and optimum, DR will increase and at temperatures higher than the optimum, DR will decrease (Atkinson and Porter, 1996; Horie et al., 2000). Therefore evaluation of the effects of warming on plant phenology requires understanding of cardinal temperatures for development and the magnitude of warming.

9.6.4.3 Effects of climate change on length of flowering period An increase in temperature due to climate change will prolong the development period of saffron. Simulation results indicate that the whole duration of flowering (flower initiation plus flower emergence) will increase by a minimum of 32 to a maximum of 38 days for each 1 C increase in mean daily temperature (Fig. 9.16A). This increment will be 9
19 days for each 1 C increase in mean daily temperature for the flower initiation period (Fig. 9.16B). On average for each 1 C increase in mean daily temperature the duration of flower initiation and flower emergence will increase by 20 and 14 days, respectively. Therefore it appears that the impact of climate change on the duration of the flower emergence period is relatively higher than for flower initiation. Simulated changes in the duration of the flower emergence period of saffron (Fig. 9.17) indicated that if the mean daily temperature increases by 1 C above the current temperature, the length of this period in all cultivation areas of the province will be longer than 50 days. The effect of warming on the phenology of plants depends on the species and geographical location (Reddy et al., 1995). In high latitudes such as in northern Europe, Canada, and Russia global warming will cause the onset of the growing season and consequently the time of occurrence of phenological stages will change (Beaubien and Freeland, 2000; Parry et al., 2004). Although most of the studies on the effects of warming on the phonological stages of plants have concerned natural vegetation (Defila and Clot, 2001), shifts in phonological stages of crop and orchard species

FIGURE 9.16 Effects of temperature elevation on (A) the length of the flowering period (sum of both phases) and (B) on the length of the flower initiation period. Solid and open circles show minimum and maximum length of the period, respectively. Simulation was conducted assuming a 0.5 C
2 C increase in mean daily temperature. From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

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FIGURE 9.17 Simulated value for duration of the flower emergence period under the condition of 0.5 C
2 C increase in mean daily temperature considering the effects of elevated temperature on DR during the first phase. From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

TABLE 9.3 Predicted dates for flower emergence of saffron under climate change condition in the main production regions of Khorasan province with mean daily temperature increase from 0.5 C to 2 C above the current conditions. Elevated temperature ( C)

0.5 1.0 1.5 2.0

Predicated date for flowering Minimum

Maximum

20 Oct 3 Nov 16 Nov 30 Nov

1 Nov 14 Nov 28 Nov 13 Dec

The values are calculated considering the effect of temperature on both flower initiation and emergence. Source: From Koocheki, A., Nassiri Mahallati, M., Alizadeh, A., Ganjali, A., 2008. Modeling the effect of climate change on flowering responses of saffron (Crocus sativus L.). Iran. J. Field Crop Res. 7, 583
594 (in Persian).

have been documented. For instance, in Germany it has been reported that with each 1 C warming, flowering for apple and cherry trees will be changed by 5
6 days. Such changes have been also reported for oats, winter rye, and sugar beet (Chmielewski et al., 2003). In lower latitudes where higher temperatures in summer already limits the growth of plants, further increase in temperature due to climate change will alter phenological processes considerably (Sivakumar et al., 2005). This has been reported for rice and wheat (Fulu et al., 2006) and also for soybean (Baker and Allen, 1993). In Iran Koocheki and Nassiri Mahallati (2016) showed that a 2 C increase in spring and summer temperatures will negatively affect the flowering pattern and time of grain filling of wheat. Since saffron flowering is highly dependent on temperature regimes, the effects of global warming will be more pronounced on this crop. Based on simulation results flower appearance and consequently the harvest date of saffron will be drastically delayed by elevated temperature in the main production areas of Iran (Table 9.3). A temperature increase by 1.5
2 C delays flowering of saffron until late November to mid-December in current production regions. Such a change in harvesting time has already been reported in the main production regions of Iran. Global warming, on the other hand, makes currently unsuitable regions due to mismatch of temperature regime more suitable for saffron production. Thus saffron cultivation areas may shift to northern latitudes. Such a shift began in Iran in the last few years.

9.7

Land suitability and zoning methodology for saffron

9.7.1 Objectives and methods of zoning Arable lands have limited capability to produce agricultural products and the limits to crop production are determined through climate and soil conditions as well as the management practices applied. On this basis productivity could be

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improved by matching crop requirements with land potentials and teaching management to overcome limitations. In this context, zoning is separation of regions with similar sets of potentials and constraints for production (FAO, 1996). The result is usually represented as a map where zones are separated using different colors or patterns. The most effective programs can then be developed for each zone, which is why the concept of zonation has appeared in agronomical research in the last four decades. Climatic zoning and its corresponding maps is the oldest category of zonation where different climates are distinguished based on relatively simple indices calculated by combining temperature and precipitation data. Biological performance of climatic zones could be improved by introducing climatic requirements and limitations of living organisms leading to bioclimatic zoning. Narrowing down for agricultural purposes, a region can be divided into agroclimatic zones (ACZs) based on homogeneity in weather variables that have the greatest influence on crop growth and yield and agroecological zones (AEZs) are defined as geographic regions with similar climate and soils for agricultural production (FAO, 1978). Van Wart et al. (2013) presented examples for different applications of agroecological zoning such as identifying yield variability and limiting factors for crop growth (Caldiz et al., 2002), selection of optimal crop management recommendations at regional scales (Seppelt, 2000), comparison of yield trends (Gallup and Sachs, 2000), determining suitable locations for new crop production technologies (Araya et al., 2010), and analyzing the impacts of climate change on agriculture (Fischer et al., 2005). Earlier approaches to AEZ involved manual overlaying of isolines representing agroclimatic information (e.g., temperature, evapotranspiration, growth degree days, length of growing period), which then was superimposed on soil resource maps (Patel, 2002). As a result, a large amount of agroecological data could not be handled easily leading to loss of information about spatial variability. Today geographic information systems (GISs) provide a fast and precise technology for integration of spatial data. With GIS, AEZ involves the representation of land in layers of spatial information (topographic, climatic, and edaphic) and combinations of these spatial layers. On the other hand, land suitability has also been defined as the fitness of a given type of land for a specified kind of land use (FAO, 1976), which in analogy is the degree of fitness of a given type of cropland for production of a given crop species. As defined by the FAO, agroecological zoning and agricultural land suitability assessment are different methods for land evaluation and therefore both schemes have more and less similar objectives (FAO, 1996). In practice, land suitability assessment for a specified crop could be performed on previously established agroecological zones with reasonable results (Fischer et al., 2012). Land suitability assessment requires models for predicting the suitability of lands for different types of agriculture. Multicriteria evaluation methods are developed for estimating suitability of lands for alternative land uses. The same methods could be applied to agricultural lands for evaluation of suitability of croplands for a given crop based on different criteria (e.g., based on climate, soil, water quality, and so on). GIS-based Multi-Criteria Decision Making (MCDM) provides facilities for incorporating MCDM in land suitability analysis. MCDM could be conducted using different methods such as ELECTRE or PROMOTHEE. However, the analytical hierarchy process (AHP) is the most commonly used method in land suitability analysis. Any MCDM method is concerned with how to combine the information from several criteria (or factors) to form a single index for suitability assessment. Sabaei et al. (2015) provided a review on different MCDM methods including AHP.

9.7.2 Application of zoning schemes to saffron While agroecological zoning and land suitability assessment are extensively used in different countries and for different crop species, application of these methods for saffron and other spices is overlooked and existing published reports suffer from lack of standard methodology. In the following, some examples are presented. Land suitability of Ardebil province (northwest Iran) for saffron cultivation was studied using 25 years of climate, soil, and topographic data and applying the MCDM
AHP method (Sobhani, 2016). The results showed that climate is the most important growth-limiting factor in the region; nonetheless, almost 40% of agricultural lands over the province are highly suitable for saffron production. Using the MCDM
AHP method Rashid Sorkh Abadi et al. (2014) showed that only 8.5% of the arable lands in Torbat (district located in central parts of Khorasan province, Iran) ranked as very suitable and 11% as unsuitable for saffron cultivation. AHP results indicated that poor water quality is the main constraint in the studied region. Rashid Sorkh Abadi et al. (2016) using the same data set of their previous study modified the initial suitability classes to fuzzy values between one and zero (one meaning very suitable and zero unsuitable) and developed a fuzzy analytic hierarchy process (FAHP). Based on the results of FAHP 43% of arable lands were shown to be very suitable for saffron production, which is significantly different from that of AHP.

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FIGURE 9.18 Comparison of saffron yield map generated from linear regression of yield and monthly average temperatures of selected districts (A) and the interpolated map of actual yield of the same districts (B). Both maps show three different zones with high spatial similarity. From Tosan, M., Alizadeh, A., Ansari, H., Rezvani Moghaddam, P., 2015. Evaluation of yield and identifying potential regions for saffron (Crocus sativus L.) cultivation in Khorasan Razavi province according to temperature parameters. Saffron Agron. Technol. 31, 1
12 (in Persian).

Any land suitability assessment requires the lower and upper thresholds of the crops for climatic, edaphic, and topographic factors used for land classification, which are usually gathered from the literature or expert knowledge. While these thresholds are fully documented for major crops (e.g., wheat and corn) for local species such as saffron they are rather subjective. On the other hand, it should be noted that the results of sophisticated methods such as AHP or FAHP are highly dependent on the quality of input data provided and even small changes in the thresholds settings may lead to considerable variation in suitability assessment. For instance, land suitability classes such as highly suitable or nonsuitable are rather descriptive notations and tell us nothing about the crop yield potential at different zones. Estimation of potential yield for each agroecological zone or land suitability class is another important problem, which is ignored in the above-mentioned studies. Coupling crop simulation models or even crop-weather models with zoning schemes provides a classification tool based on crop yielding ability (Caldiz et al., 2002). In a zonation study, Kouzegaran et al. (2014) developed a multiple regression model for prediction of saffron yield from monthly temperature and relative humidity for selected districts in Southern Khorasan province, Iran. The model was then used as a yield prediction tool over the province and the resulting yield was spatially interpolated with GIS for mapping different zones based on saffron yielding ability. Using 20 years of data Tosan et al. (2015) developed three separate regression models based on saffron yield and monthly average, monthly minimum, and monthly maximum temperatures in selected districts over Khorasan Razavi province, Iran. These regression models were then used to generate three yield maps where zones were separated according to saffron yield. Comparison of these maps with the maps of actual yields of saffron over the province indicated that yields predicted using monthly average temperatures had the highest accordance with actual yields (Fig. 9.18A and B). Performing a agroecological zoning scheme at large scale (e.g., province) is the best method for separating homogeneous arable lands. The suitability of any crop species including saffron can then be assessed within each zone based on the soil and climate factors used for zoning. Estimating the potential yield of saffron in homogeneous zones using simulation models is required to provide a quantitative measure for the qualitative land suitability classes.

9.8

Conclusion

In this chapter different types of models from simple crop-weather regression to more sophisticated dynamic models with examples of their applications in saffron are reviewed. Overall, application of modeling approaches within saffron research is rather new. The results presented in this chapter show that modeling studies on saffron have a short history, starting in 2008, but progress is promising. Unsurprisingly most of these studies were performed in Iran, the world’s highest saffron-producing country. Undoubtedly, compared to regression models dynamic simulation models are more powerful tools for quantifying the complex interactions between crop growth and development with environmental factors. However, development of such models is highly dependent on experimental data, which are so far lacking for saffron. Among different applications, simulation models have two important objectives: namely, prediction of crop yield and optimization of

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management practices. The results of existing modeling attempts presented in this chapter show that when the required data for development of dynamic models are not available, simple models can work as a reasonable alternative. Multiple regression models and artificial neural networks could be applied for saffron yield prediction at district or even province scales, and response surface modeling is an appropriate approach for optimization of management practices (e.g., fertilizers, irrigation).

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Schlenker, W., Roberts, M.J., 2009. Nonlinear temperature effects indicate severe damages to U.S. crop yields under climate change. Proc. Natl. Acad. Sci. U.S.A. 106, 15 594
15 598. Sepaskhah, A.R., Amini-Nejad, M., Kamgar-Haghighi, A.A., 2013. Developing a dynamic yield and growth model for saffron under different irrigation regimes. Int. J. Plant Prod. 7, 473
504. Seppelt, R., 2000. Regionalised optimum control problems for agroecosystem management. Ecol. Model. 131, 121
132. Sinclair, T.R., Muchow, R.C., 1999. Radiation use efficiency. Adv. Agron. 65, 215
265. Sivakumar, M.V.K., Das, H.P., Brunini, O., 2005. Impacts of present and future climate variability and change on agriculture and forestry in the arid and semi-arid tropics. Climatic Change 70, 31
72. Smith, A.E., Mason, K., 1997. Cost estimation predictive modeling: regression versus neural network. Eng. Economist 42 (2), 137
161. Smith, W.J., 1914. The effect of weather upon the yield of corn. Monthly Weather Rev. 42, 78
93. Sobhani, B., 2016. Agroclimatic zoning of saffron in Ardabil province using AHP method. J. Saffron Res. 4, 72
86 (in Persian). Steduto, P., Hsiao, T.C., Raes, D., Fereres, E., 2009. AquaCrop-the FAO crop model to simulate yield response to water: I. Concepts and underlying principles. Agron. J. 101, 426
437. Stewart, D.W., Dwyer, L.M., Carrigan, L.L., 1998. Phenological temperature response of maize. Agron. J. 90, 73
79. Sto¨ckle, C.O., Kemanian, A.R., 2009. Crop radiation capture and use efficiency: a framework for crop growth analysis. In: Sadras, V., Calderini, D. (Eds.), Crop Physiology: Applications for Genetic Improvement and Agronomy. Academic Press/Elsevier, Amsterdam, pp. 145
170. Tannura, M.A., Irwin, S.H., Good, D.L., 2008. Weather, Technology, and Corn and Soybean Yields in the U.S. Corn Belt. University of Illinois at Urbana
Champaign Dept. of Agricultural and Consumer Economics Marketing and Outlook Res. ,http://www.farmdoc.illinois.edu/marketing/ morr/morr_08-01/morr_08-01.pdf.. Tosan, M., Alizadeh, A., Ansari, H., Rezvani Moghaddam, P., 2015. Evaluation of yield and identifying potential regions for saffron (Crocus sativus L.) cultivation in Khorasan Razavi province according to temperature parameters. Saffron Agron. Technol. 31, 1
12 (in Persian). Van Delden, A., 2001. Yielding Ability and Weed Suppression of Potato and Wheat Under Organic Nitrogen Management (Ph.D. thesis). Wageningen University, The Netherlands, p. 197, ISBN 90 5808 519/8. Van Ittersum, M.K., Leffelaar, P.A., Van Keulen, H., Kropff, M.J., Bastiaans, L., Goudriaan, J., 2003. On approaches and applications of the Wageningen crop models. Eur. J. Agron. 18, 201
234. Van Ittersum, M.K., Cassman, K.G., Grassini, P., Wolf, J., Tittonell, P., Hochman, Z., 2013. Yield gap analysis with local to global relevance-A review. Field Crop Res. 143, 4
17. Van Laar, H.H., Goudriaan, J., Van Keulen, H., 1997. SUCROS97: Simulation of Crop Growth for Potential and Water-Limited Production Situations. Quantitative Approaches in Systems Analysis, No. 14. C.T. de Wit Graduate School for Production Ecology and Resource Conservation, Wageningen. Van Wart, J., Van Bussel, L.G.J., Wolf, J., Licker, R., Grassini, P., Nelson, A., et al., 2013. Use of agro-climatic zones to upscale simulated crop yield potential. Field Crop Res. 143, 44
55. Wallach, D., 2006. Evaluating crop models. In: Wallach, D., Makowski, D., Jones, J.W. (Eds.), Working with Dynamic Crop Models. Elsevier, Amsterdam, pp. 11
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614. Yan, W., Wallace, D.H., 1998. Simulation and prediction of plant phenology for five crops based on photoperiod by temperature interaction. Ann. Bot. 81, 705
716. Yin, X., Kropff, M.J., McLaren, G., Visperas, R.M., 1995. A nonlinear model for crop development as a function of temperature. Agric. For. Meteorol. 77, 1
16.

Further reading Shaykewich, C.F., 1995. An appraisal of cereal crop phenology modeling. Can. J. Plant Sci. 75, 329
341.

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Chapter 10

Saffron crop protection Mohammad Bazoobandi, Hasan Rahimi and Mahmud-Reza Karimi-Shahri Department of Plant Pests and Diseases Protection, Khorasan Razavi Agricultural and Natural Resources Research Center, Mashhad, Iran

Chapter Outline 10.1 Introduction 10.2 Weeds 10.2.1 Weed and saffron ecophysiology 10.2.2 Weed presence in saffron fields and possibility of weed control 10.2.3 Critical period of weed control 10.2.4 Dominant weeds in saffron fields 10.2.5 Weed management 10.3 Pests 10.3.1 Mites (Acari) and Insects (Insecta)

10.1

169 169 170 170 171 171 174 176 177

10.4 Pathogens 10.4.1 The fungal agents of saffron corm rot 10.4.2 Fusarium rottings of the saffron corm 10.4.3 Nematodes 10.4.4 The saffron pathogenic bacteria 10.4.5 Saffron viruses 10.5 Conclusion References Further reading

182 182 182 183 183 183 184 184 185

Introduction

Saffron, similar to other agricultural products, is disturbed by some harmful biotic factors. The effects of these biotic factors depend on the harmful species, climatic factors, farm management, and so forth, which all are potential means of exerting considerable damage to the crop. On the other hand, there are useful biotic organisms that feed on the harmful biotic factors and play a significant role in the natural control of saffron pests. As with other crops, weeds also interfere in saffron growth and production. Weeds’ intervention occurs mostly through competition for growth resources such as water, nutrients, space, and atmospheric gases. Moreover, allopathic adverse compacts of weeds should not be neglected. Weeds also provide a suitable environment for various pests and diseases in saffron fields. Among the different pests that restrict saffron production, weeds rank first. Depending on weed density, species, and management, saffron yield and quality losses may differ between locations (Bazoobandi and Vojoudi, 2016). Plant pathogens cause loss of function of the plant and damage the agricultural products. Saffron shows low sensitivity to live environmental stressors (i.e., plant pathogens) due to the special climatic factors present in the areas that saffron is able to grow. Therefore, live plant pathogens invade saffron less frequently and when they do invade can be managed through adherence to agricultural principles and farm hygiene. Fungal agents are an additional category of saffron pathogens. Weeds, pests, and diseases of saffron are discussed in the following sections.

10.2

Weeds

Several issues make it difficult to have a satisfactory weed control program in saffron fields. Factors restricting weed management in saffron are as follows: 1. Land remains occupied by saffron for several years, making it possible for perennial weed species to extensively develop. 2. Due to the short and narrow shaped leaves of saffron, it cannot efficiently compete with weeds for light (EslamAbbasi, 1996). 3. Corms are placed at 20 cm soil depth, making cultivation practices to control weeds difficult to carry out. Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00010-1 © 2020 Elsevier Inc. All rights reserved.

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4. Herbicide application is not conventional as their residue in the crop is a major concern. Moreover, no herbicide is officially registered for saffron. 5. Hand weeding is very costly. Eighty laborers are needed for hand weeding of one hectare. Therefore, weeds are often left to grow almost throughout the year except during flowering (Bazoobandi and Vojoudi, 2016). Keeping in mind the problems that were discussed earlier, there is intense competition between weeds and saffron from late winter to early spring, coinciding with the peak of saffron vegetative growth. Such a competition results in reduction of size and nutrient deposit of corms, which in turn affects flowering in the succeeding year.

10.2.1 Weed and saffron ecophysiology Efficient weed management requires understanding of the ecophysiology of saffron and weeds. Saffron biomass partitioning has been investigated. It has been shown that total corm dry weight decreases up to the 120th day of plant growth and then increases until the end of the lifecycle (mid-May). However, different behavior may be observed in the case of root and leaf dry weights. These two indices usually increase up to the 120th and 150th days of the plant growth cycle, respectively, followed by decreasing trends. Therefore, early during the growing season, leaves and root systems develop using mother corm reservoirs, but at the end of the growth cycle, replacement corms grow by translocation of reservoirs from other saffron organs. The number and dry weight of buds increase during the first 160 days of the growing season. The total length of roots and leaf area increase up to the middle of the growth cycle and then decrease. There is a rapid increase in saffron flower yield during the first phase of the flowering period and then a decline with a slower tendency during the second flowering phase (Behdani et al., 2016). A schematic of saffron growth stages is shown in Fig. 10.1. Different weed management strategies may be applied based on saffron tolerance to mechanical and chemical methods as well as on which weeds are present.

10.2.2 Weed presence in saffron fields and possibility of weed control Weed presence in saffron fields may be investigated over four steps: 1. From first irrigation to flower emergence of saffron: Corm emergence takes about 20 days after irrigation, which provides a good opportunity for those weeds that have their seeds close to the soil surface to rapidly germinate and emerge.

FIGURE 10.1 Growth stages of saffron. From Bazoobandi, M., Vojoudi, H., 2016. Saffron Integrated Weed Management. Tohid-Manesh Press, Neyshabour, Iran (in Persian).

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FIGURE 10.2 Leaf area index trend during the (A) first and (B) second growing season. From Mirhashemi, S.M., Bannayan, M., Nezami, A., Nassiri-Mahallati, M., 2015. Evaluation of the extinction coefficient, radiation absorption and use efficiency of saffron (Crocus sativus L.). Saffron Agron. Technol. 3 (3), 203
216 (in Persian).

2. During saffron flowering: This period contains optimal growth conditions for weeds. Flowers of saffron emerge prior to its leaves. Early irrigation and nitrogen application may change this usual phenomenon; during this period weeds have the chance to grow freely. 3. During saffron vegetative growth stage: Following flower harvesting (generative stage), vegetative growth starts and continues to the time of corm inactivity in mid spring. Saffron canopy development is minimal during this period especially within the first year of field establishment. Leaf area index (LAI) is approximately one, which is very small compared to many other crops. Maximum LAI in the first year is less than 0.5. It may reach to about 1.8 in the second year (Mirhashemi et al., 2015). Leaves are very narrow in shape, and veins constitute the major part of leaf. Veins lack chlorophyll (Kafi et al., 2006). All these factors make saffron an inefficient competitor for light (Fig. 10.2). 4. From corm inactivity to next flowering: Duration of this period is about 6 months from early May to early October. Leaves turn to yellow and corms become inactive in May. Various types of weeds, including annuals and perennials, grow easily in the absence of saffron and remove soil nutrients and moisture. Different weed management methods, which may be implemented in saffron fields according to its growth stage and other cultivation practices, are summarized in Table 10.1.

10.2.3 Critical period of weed control As defined, “the critical period for weed control (CPWC) is a period in the crop growth cycle during which weeds must be controlled to prevent yield losses” (Knezevic et al., 2002). No investigation has been carried out to find this period in saffron; however, observing saffron phenology, it may be concluded that the last 2 months of the vegetative growth stage, when daughter corms form on mother corm (Fig. 10.1), can be considered as the CPWC. Proper development of new corms ensures acceptable yield in the next year. Increases in temperature and relative humidity favors optimum growing conditions for weeds during these 2 months (Eslam-Abbasi, 1996).

10.2.4 Dominant weeds in saffron fields Weed identification and surveillance are necessary for a sustainable weed management program. Except wild saffron, which is the only crop bound weed in saffron, other weeds are season bound and may be found in any other crop. Different types of weed species can be found in saffron fields throughout the year. Out of 184 weed species from 33 families and 128 genera in south Khorasan, one of the major areas of saffron production, 113 species were annuals and 71 were perennials. Asteraceae, Fabaceae, Poaceae, and Brassicaceae ranks first to fourth, respectively (Kafi et al., 2006). Less variation has been reported in higher latitudes such as Mashhad (Padarloo, 2007). Fall-germinating weeds such as Descurainia sophia, Papaver rhoeas, and Lolium rigidum are the most feared species by farmers in Spain (Marı´, 2010). The major weeds in saffron fields in Kashmir are Euphorbia helioscopia, P. rhoeas, Lepidium virginicum,

TABLE 10.1 Weed management according to saffron phenology and cultivation practices. Month

June

July

August

September

October

Growth stages

Dormancy

Leaf formation

Flower initiation

Initial growth

Development

Cultivation Practices

Corm planting

Mechanical weed controls

Harrow/ hand weeding

Chemical weed controls

December

January

First irrigation

Second irrigation

March

Harrow

N Fertilizer

Micro nutrients spray

Harrow/ hand weeding

Harrow

Barley cover crop

Glyphosate

February

Middle growth

April

May

Final growth

Growth deactivation

Third irrigation

Fourth irrigation

Harvest Farm yard manure

Ecological weed controls

November

Barley removal

Selective herbicides

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Salvia moorcroftiana, Chonspora tanella, Galium tricorne, Tulipa stellata, Erodium cicutarium, Lithospermum arvense, Ranunculus arvensis, Medicago lupilina, Filago arvense, Poa bulbosa, Crepis saneta, D. sophia, Polygonum aviculare, and Chenopodium album, among others (Pir et al., 2008).

10.2.4.1 Annual winter weeds The seeds of this group of weeds germinate subsequent to the first irrigation in fall and the weeds remain as rosette/tiller during winter. Flowering and seeding occurs in early spring. Examples of these species are Lepidium draba Desv., Gallium aparine L., Silene conoidea L., and Alopecurus myosuroides Hudson.

10.2.4.2 Annual summer weeds These species cannot tolerate temperatures below 0 C. Seeds germinate in late winter as soil temperature increases, which coincides with maximum vegetative growth of saffron. E. helioscopia L., Heliotropium europaeum L., Portulaca oleracea L., Chrozophora tinctoria L., C. album L., Avana fatua L., and Setaria viridis L., are among these groups.

10.2.4.3 Perennials Simple perennials withstand winter with the help of their roots and regenerate through seeds. These weeds continue their growth and development during spring and summer, when the saffron corm is not active. Foliar senescence and death occurs in the fall season when temperature decreases. Cichorium intybus L., Malva neglecta Wallr., Plantago lanceolata L., Taraxacum iranicum V. Soest, and Tragopogon Persicus Baiss. are important species in this group. Creeping perennial weeds are the most noxious weeds in the world. These species not only produce seeds, but also reproduce through other asexual parts like rhizomes, corms, and so on. Alhagi spps., Cirsium arvense L. Scop., Sophora alopecuroides L., Cynodon dactylon L., and Pers. Agropyrum repens L. are major infecting species in Iranian saffron farms. A threatening species named Moraea sisyrinchium Ker Gawl. (syn. Gynandriris sisyrinchium), the Barbary nut, whose corms closely mimic saffron plants, has been identified in saffron fields and should be closely monitored. Compared to saffron, leaves of Barbary nut are wider and its corms are brighter in color (Fig. 10.3).

FIGURE 10.3 Leaves, corm, and flowers of Moraea sisyrinchium infecting saffron fields. From Bazoobandi, M., Vojoudi, H., 2016. Saffron Integrated Weed Management. Tohid-Manesh Press, Neyshabour, Iran (in Persian).

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10.2.5 Weed management Despite thousands years of hand weeding, plowing, and 50 years of herbicide application, farmers still have a great challenge with weeds. About half of human resources are being spent for hand weeding in small farms all over the world. Therefore, weeds impose a socio-economic impact on saffron farming. A sustainable approach to weed management requires a change in our view towards weeds (i.e., living with weeds rather than their elimination). Logical implementation of different weed control methods including chemical control is recommended (Rashed-Mohassel et al., 2001).

10.2.5.1 Monitoring Geostatistical techniques may be used to describe spatial distribution of saffron weed populations. The semivariogram analysis indicated that more than 50% of the variation of sample density is due to spatial dependence, which suggested that most of the species are patchy. Investigations have shown that semivariogram parameters did not change significantly over time for weeds such as Cardaria draba, which indicated the relative stability of patches of these weeds. Weed management tools can change weed distribution overtime. A Hordeum spontaneum population has been affected by postemergence weed control and its spatial correlation decreased from 79.4% to 73.1%. The spatial correlation of C. draba and Carduus pycnocephalus increased after postemergence control. Patchy weed distribution offers large potential for using site specific weed control in the field (Makarian et al., 2008).

10.2.5.2 Prevention Avoiding application of unprocessed farm yard manure (FYM): Application of FYM is very conventional in saffron cultivation. Unprocessed FMY contains a large number of weed seeds. It is estimated that about 25% of seeds in livestock hay remain intact and appear in the livestock excretion (Davis et al., 2008). FMY solarization for 2 months is recommended to reduce the weed seed viability (Salimi et al., 2008). Escape grazing: Sheep grazing has been reported to control weeds efficiently (Zare-Hosseini et al., 2014); however, as already mentioned, their excretion would transfer many weed seed. Soil cleansing from corm: When establishing new farms, corms are transferred from other fields, which may be infected with weed seeds. Therefore, it is necessary to clean attached soil from around corms as much as possible. Weed control at field margins and irrigation channels: Weeds that grow by the side of saffron fields and irrigation channels can easily be controlled by nonselective herbicides.

10.2.5.3 Mechanical control Hand weeding: Fields should be hand weeded prior to flowering. Better weed control can be achieved when weeds are younger. The number of human laborers for hand weeding is estimated to be 80 per hectare. The labor market is expensive. The first hand weeding is carried out following flower harvesting (i.e., after the second irrigation). The second weeding, if required, would be carried out 1 month later to interrupt winter weed establishment. Two more subsequent weedings may be implemented after saffron foliar harvesting and in summer, respectively. Breaking soil crust: Various small breaking machines are commonly used in saffron farms after the first irrigation (Bazoobandi and Vojoudi, 2016).

10.2.5.4 Soil solarization The soil solarization method takes advantage of sun light to create high temperatures under plastic sheets, which destroys weed seeds and any other living vegetation. The moist soil surface is covered by transparent plastic sheets for 6 weeks. Clear and sunny weather is needed to increase the temperature over 65 C (Rahimi, 2016b).

10.2.5.5 Mix cropping Mix cropping is one nonchemical weed control method (Khoze Mary, 2000). Different crops such as barley and pulses can be sown in mixture with saffron to reduce weed competition. Pulse crops also support nitrogen fixation. Koocheki et al. (2009) showed that spring barley had a higher relative advantage compared to winter barley in mix cropping with saffron.

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FIGURE 10.4 Effects of crop residues and cover crops on (A) weed dry weight and (B) weed density of broad leaf and narrow leaf weeds in a saffron field. From Shabahang, J., Khorramdel, S., Amin-Ghafori, A., Gheshm, R., 2013. Effects on management of crop residues and cover crop planting on density and population of weeds and agronomical characteristics of saffron (Crocus sativus L.). J. Saffron Res. 1 (1), 57
72 (in Persian).

Farmers even sow saffron in almond farms. It has been reported that the total relative advantage of saffron mix cropping with spring wheat, chickpea, and cumin (Cuminum cyminum L.) were all higher compared to single cropping. However, saffron yield was decreased in mix cropping (Farhoudi et al., 2003; Naderi-Darbaghshahi et al., 2009). Research has shown that Vicia faba inclusion in mix cropping with saffron could reduce weed density by 93%, which resulted in 10.1 kg ha21 flowers (i.e., a 540% increase compared to weed infested plots) (Shabahang et al., 2013) (Fig. 10.4). It may be concluded that cover cropping could increase soil nitrogen availability. Applying different crop residues and cover crops decreased the density and dry weight of weeds via allelopathy. Cover crops increased growth and yield of saffron due to enhancement of nutrient and nitrogen availability (Fig. 10.5).

10.2.5.6 Cover crops The cover crop barley significantly decreased weed dry matter weights. Barley cultivation resulted in the lowest weed dry matter weight, which was similar to hand weeding. In conclusion, the treatment with cover crops showed the best performance in weed control and saffron yield compared to other studied weed management methods (Zare-Hosseini et al., 2014).

10.2.5.7 Chemical control (Herbicides) Traditionally there is no interest in the use of herbicides in saffron fields; however, due to the high cost of human labor, herbicide application is unavoidable. Few herbicides have been applied in saffron and only some of these have been documented.

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FIGURE 10.5 The effects of crop residues and cover crops on (A) flower number and (B) stigma dry weight of saffron. From Shabahang, J., Khorramdel, S., Amin-Ghafori, A., Gheshm, R., 2013. Effects on management of crop residues and cover crop planting on density and population of weeds and agronomical characteristics of saffron (Crocus sativus L.). J. Saffron Res. 1 (1), 57
72 (in Persian).

1. Graminicides: Haloxypfop-R-methyl EC 1.8% (1 L  ha21) and oxyfluorfen EC 24% (2 L  ha21) were recommended to control narrow leaved weeds (Nurbakhsh, 2016). Cycloxydim is another recommended graminicide (Mousavi et al., 2013). However, there are reports of adverse effects of this herbicide on stigma yield and leaves of saffron (Zare-Hosseini et al., 2014). 2. Broad-leaved herbicides: Metribuzin WP 70% is one of the few herbicides that farmers apply after flower harvesting to control broad leaved weeds in saffron fields without any injury symptoms (Norouzzadeh et al., 2007). Although tribenuron methyl (20 g  ha21) has been recommended (Nurbakhsh, 2016), if applied while leaves are green and active, it would destroy the whole corm within a few weeks after application. Glyphosate may be applied prior to land preparation (Abbaspoor et al., 2011). Ioxynil EC 22.5% is another applicable herbicide in saffron farms. (Abasian et al., 2013).

10.3

Pests

Various pests attack saffron fields; however, mites are the group with greatest impact. The most important pests of saffron are listed below.

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10.3.1 Mites (Acari) and Insects (Insecta) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Rhizoglyphus robini Claparede, 1869 (Acari: Acaridae) Tyrophagus putrescentiae Schrank, 1871 (Acari: Acaridae) Petrobia latens Muller, 1776 (Acari: Tetranychidae) Brgobia praetiosa Koch, 1835 (Acari: Tetranychidae) Penthaleus major Duges, 1834 (Acari: Penthaleidae) Agriotes sp. (Coleoptera: Elateridae) Thrips tabaci Lindeman, 1889 (Thysanoptera: Thripidae) Collembolothrips sp. (Thysanoptera: Thripidae) Haplothrips reuteri karny, 1907 (Thysonoptera: Phlaeothripidae) Myzus certus walker, 1849 (Homoptera: Aphididae) Myzus persicae Sulzer, 1776 (Homoptera: Aphididae) Aulacorthum palustre HRL. 1947 (Homoptera: Aphididae) Xenylla boerneri Axelson, 1905 (Collembola: Hypogastruridae) Sphaeridia pumilis Krausbauer, 1898 (Collembola: Sminthuridae)

R. robini mite, the short-tailed bandicoot rat, and the southern mole volefor are discussed in this chapter because they are more common pests. They cause significant damages to saffron through feeding on saffron corms, which leads to decay, destruction, and eventually thinning saffron farms.

10.3.1.1 Rhizoglyphus robini Claparede, Acari: Rhizoglyphidae The Bulb Mite, R. robini Claparede, is one of the most common tuber plant pests. Rahimi and Kamali (1993) for the first time reported this pest in saffron fields in Gonabad and Qaen, Iran. This mite can be found in all cities where saffron is planted. The level of contamination is varied depending on the time of the start of the first cultivation and treatment considerations in different regions (Rahimi and Kamali, 1993). Bulb Mites have been recorded feeding on Iris, Lily, Narcissus, Gloriosa, Hippeastrum, Eucharis, Orchid, Hyacinth, and Tulip bulbs, Dahlia tubers, as well as Freesia and gladiolus corms. These mites also infest vegetable bulbs (Manson, 1972). 10.3.1.1.1

Morphology and biology

R. robini have an oval body shape and are translucent with a shiny appearance and are mostly sedentary. Their legs are short and reddish brown with a significant amount of hair (Fig. 10.6). They have different forms. There are two kinds of males: homomorphic and heteromorphic. The life duration of this pest lasted 13.75 days under in vitro conditions where temperature was held at 25 C 6 1 C, with saturated relative humidity, and full darkness inside a cage with growing conditions on the slices of saffron corms (Rahimi and Kamali, 1993). The pests are active throughout the year and can produce several generations. However, because of the suitable conditions (i.e., humidity and temperature) in spring and autumn, the population is significant during these seasons. The population is heavily reduced because of the heat and dryness of the soil during summer and the cold weather during winter. Their activities are similar to other destructive agents and depend on three factors: humidity, temperature, and food. In the absence of any of these three factors, their activities do not perpetuate. In normal conditions of summer, out of these factors, the lack of moisture around the corms leads to a significant reduction in their populations. Summer irrigation artificially creates humidity around the saffron corm. This situation along with other factors including available food (saffron corms) and temperature will form the pest activity triangle. If saffron fields are irrigated during the summer (period of rest of saffron) for any reason, the conditions would become appropriate and rapid development of the mite population would occur (Rahimi et al., 2008). 10.3.1.1.2 Types of damage By providing the R. robini living conditions, which were already mentioned, the corms of saffron may be attacked by the pest often at scars and sometimes at healthy parts of the plant. Moreover, while feeding and tunneling inside the corm, the Bulb Mite begins to multiply and creates dark spots in the corm. The holes will gradually expand and infective agents will easily penetrate from scars into the corm and hasten decay in the corm (Fig. 10.6). Different stages of their lifecycle are visible with binocular. Plants that are attacked by the pest have thinner and shorter leaves than healthy ones. The leaves of infected plants become yellow more readily than usual. Deficiencies are observed in

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FIGURE 10.6 (A) Damage of bulb mite on saffron corm and (B) Bulb Mite, R. robini. From Rahimi, H., Kamali, K., 1993. Laboratory studies on biology of bulb mite Rhizoglyphus robini (Acari: Acaridae) and its damages on saffron corm in Gonabad and Qaen. Sci. J. Agric. 16, 53
63 (in Persian).

severely infected plants such as fading petals and shorter stigmas. The total damage after several years is the thinning of saffron fields.

10.3.1.1.3 Distribution of Rhizoglyphus robini mites Mite infestations have been faced in all cities of Khorasan-Razavi province, Iran where saffron is grown including Gonabad, Bajestan, Khaf, Torbat-e-Heydarieh, Zavaeh, Nasrabad, Kashmar, Bardaskan, Mahvelat, Khalilabad, Roshtkhar, Mashhad, Torbat-e-Jam, Fariman, Nishabur, Sabzevar, and Chenaran. Over the last few years due to summer irrigations in areas with adequate water sources, mite pollution has risen, which can be readily seen in Torbat-eHeydarieh, Zavaeh, Nasrabad, Torbat-e-Jam, and Chenaran.

10.3.1.1.4

Factors causing Rhizoglyphus robini to be problematic

Prevailing various factors may provide suitable conditions for R. robini to be a problem in saffron production. These factors may be listed as: 1. 2. 3. 4. 5. 6. 7. 8. 9.

saffron cultivation without adopting the principles of sustainable planting increasing summer irrigation and providing favorable conditions for R. robini mites saffron cultivation in higher latitudes where climate does not favor saffron lack of careful supervision and proper isolation during saffron transfer harvesting and replanting old corms lack of technical and hygienic considerations in transferring corms from source to destination for planting failure to adhere to correct planting depth lack of knowledge of planting and harvesting over use of chemical and manure fertilizers

10.3.1.1.5 Technical strategies to prevent and control saffron mites In terms of botany, saffron is an annual plant and every year it corms renew and old corms (mother’s corm) die. However, renewed corms last several years, often 7
8 years and sometimes even longer in the soil. This long period provides an opportunity for harmful agents to increase to a critical level. For this reason, to reduce the damage by pests, measures should be taken in two categories: prevention and control of pests in old farms and prevention and control of pests in construction of new farms.

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10.3.1.1.6

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Prevention and control of mites in old farms

Intense mite activities have been observed in farms where: (1) polluted corms are planted, (2) the soil texture is hard, (3) planting depth is lower than usual, (4) irrigation occurs during summer, and (5) precipitation occurs during summer. To reduce damage in these farms, effective management factors include: 1. Avoid summer irrigation: Because it artificially creates humidity around the saffron corms and promotes conditions appropriate for mite growth and infective agents in the soil. This in turn will eventually lead to the spoilage and decay in saffron corms (Rahimi et al., 2008). 2. Summer rainfall: Because of rainfall during the summer in higher latitudes, a similar effect to summer irrigation can be observed. Even more destruction may occur due to flood, which could harm saffron corms. Therefore, for successful cultivation of saffron, climate is very important. Production is specified to subtropical areas with relatively cold winter and dry summer with a minimum of 215 C and a maximum of 35 C
40 C with no precipitation in the summer. 3. Soil texture: Since heavy soils retain more moisture, they provide better conditions for mites. It is therefore recommended to add sand to heavy soils to make the soil less heavy. Adding sand to the soil should be done in late summer prior to saffron field irrigation. 4. Planting depth: Fields with planting depth of less than usual are more susceptible to destructive factors such as extreme heat and drought in summer and frost in winter. It is recommended to reduce damage in late summer (late period of saffron rest) by adding light soil to make planting depth greater than 15 cm. 5. Strengthening saffron: Strengthening saffron by spraying soluble fertilizers on leaves in February and March leads to better results. Despite existence of mites, saffron will grow better and the next year’s yield would be less affected by mite damage. 6. Irrigation: Irrigation during fall and winter seasons is very important. Although precipitation during these seasons reduces the need for irrigation, there is not enough rain to meet saffron demands. Hence, three rounds of irrigation are essential (i.e., prior to flowering, after flowering, and the last irrigation when the leaves fall down in April). In the absence of winter precipitation, irrigation should also be considered. 10.3.1.1.7

Prevention and control of mites prior to corms planting

1. Land selection: Selected land for the cultivation of saffron should be fertile and rich in nutrients and organic matter. At the same time, a light soil texture is needed for proper ventilation. 2. Fertilization: Fertilization before planting is necessary. Manure (cow manure) free of weed seeds is very appropriate. 3. Amendment of farms that have a long history of cultivation: Saffron farms lose their efficiency for farming due to frequent cultivation. Therefore, they should be amended. Solarization at the beginning of summer for 30 days is recommended (Rahimi et al., 2013). 4. Corms: Select corms for planting from dry soil. Corms prepared from irrigated land should be strictly avoided. 5. Isolation and selection of healthy corms for planting: Before transferring corms, healthy corms free of dark spots should be selected. 6. Transferring corms: At the time of transfer from the origin to the destination, healthy corms should be separated by net bags or boxes suitable for packaging and be rapidly transferred and planted immediately. It is necessary that quarantine authorities approve the transfer of corms. Moreover, bulk transfer to a region should be avoided. 7. Corm disinfection: Since disinfecting reproductive parts (i.e., corms) is one of the important principles of sustainable agriculture, it is necessary to disinfect corms with fungicides and acaricides before planting. It is better for disinfection to be performed by spraying dissolved disinfectant and avoiding floating corms in the toxic solutions. 8. Planting: Planting corms in rows with a distance of 20
25 cm between rows and 5 cm within the row is suggested to allow enough space and light interception. Mass planting is not recommended because fillings and condensations are more likely to take place. This prevents sufficient growth and destructive agents are more readily to expand. 9. Planting depth: Planting depth of 18
20 cm is recommended depending on soil texture. Rahimi et al. (2008) recommended 20 cm depth of sowing to have higher yield and smaller mite populations. 10. The number of corms per unit area: The number of corms should be high enough per unit area to reach economical harvest in a shorter time so that mites have less opportunity to increase their population. 11. Planting time: The best time for saffron planting to increase yield while reducing damage is June. Rahimi (2016a,b) stated that the yield of saffron cultivation in June is more than in September. Comparing two planting dates

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(June and September), Koocheki et al. (2016) observed more leaf area and saffron yield with the spring planting date. 12. Irrigation after planting: Irrigation should not be implemented immediately after planting. It would increase mite population and reduce saffron performance (Rahimi, 2016a,b). 13. Saffron nutrient requirement: Spraying nutrients on saffron leaves during growing seasons, especially in February and March, enhances its performance and makes plants more tolerant against destructive agents.

10.3.1.2 Harmful rodents in saffron fields Several rodents feed on corms, which can cause irreversible damage. The short-tailed bandicoot rat, the southern mole vole, porcupines, and sometimes rabbits can be mentioned as the most important rodents. 10.3.1.2.1 The short-tailed bandicoot rat (Nesokia indica Gray) The short-tailed bandicoot rat exists in many saffron cropping areas. They feed on corms and dig tunnels. Significant damage occurs especially during the summer. The short-tailed bandicoot rat is generally brown on the upper parts and lighter on the underside. It has long, dense, and soft hair in the winter, but the hair is short, sparse, and stubbly in the summer. The broad feet and the tail are scantily haired. It length is between 14 and 20 cm (Fig. 10.7). The short-tailed bandicoot rat spends most of its time in a burrow, which comprises many tunnels and chambers. The depths may be up to 60 cm and the burrow may be up to 9 m long, covering an area of up to 120 m2. One chamber is lined with vegetation for nesting. A big rat’s nest embankment at the entrance to the exit can be seen in Fig. 10.7. The rat is active throughout the year and causes severe thinning of saffron corms on farms. Moreover, the burrows of the rat lead to waste of irrigation water (Shahrokhi et al., 2002). 10.3.1.2.2 The southern mole vole (Ellobius fuscocapillus Blyth) Moles are small mammals rarely taller than 15 cm in length. They have cylindrical bodies, velvety fur, very small, inconspicuous ears and eyes, a short tail with length of 2 or 3 cm, reduced hindlimbs, and short, powerful forelimbs with large paws adapted for digging (Fig. 10.8). This animal can be found in most countries. However, it cannot be seen easily due to its underground life. The mole with its underground activities feeds on different plants. It is active throughout the year close to mountainous areas. Thus, saffron fields in mountainous areas are heavily attacked by the mole. Most mole damages, in addition to feeding on saffron and thinning of farms, also waste irrigation water (Shahrokhi et al., 2002).

FIGURE 10.7 The short-tailed bandicoot rat. From Rahimi, H., 2016a. Saffron Pests (Survey and Management). Sokhan-Gostar Press, Mashhad, Iran (in Persian).

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FIGURE 10.8 The southern mole vole. From Rahimi, H., 2016a. Saffron Pests (Survey and Management). Sokhan-Gostar Press, Mashhad, Iran (in Persian).

10.3.1.2.3 Preventing and controlling rats in saffron fields 1. Hedges and stones at the borders of saffron fields should be limited as much as possible to reduce shelters for rats. This will reduce rats’ reproduction and other predators will catch them easier. 2. The borders or bridges within the boundaries of saffron fields provide good places for their nesting. Since the bottom of their nest is higher than the static level of water, after irrigation their nests do not fill with water, which prevents them from drowning. Because of this, when planting and establishing a saffron farm, nesting places within borders of the farm should be limited as much as possible. 3. To control rats, poisons or poisoned baits can be used. For this purpose, first one must destroy all of the nests at the farm by crushing the nests. The day after crushing, the nests that remain open indicate rats are active there and should be treated with poison, poisoned baits, or any other suitable means of extermination. To control rats, poison gases such as tablets that sublimate gaseous pesticides are used. These poisons are often placed in protective coatings. Tablets emit their poisonous content within half to one hour. During the summer, because the farm is dry and humidity in the saffron fields is low, the sublimation process is very slow and desirable effects in controlling rats will not be met. Therefore, to enhance the process after the diagnosis of active nests, it is necessary to first insert about half of a liter of water to speed the sublimation process. Then immediately put tablets inside the nest. The nest entrance should be blocked so gas does not leak out. If poisoned baits are supposed to be used against rats, it will be better to select baits that are preferable for rats depending on the seasons of the year. For instance, appropirate winter baits are nutrient rich and energetic, and appropriate summer baits are juicy and sweet. Gloves should be used when preparing the bait. If the bait is soaked in animal fat and prepared in aluminum wrappers, it will be more efficient. Because of the strong smell, rats will be encouraged to use it and because of the wrappers, the bait will last longer. The bait must be poisoned by pesticides that do not cause immediate death of the rats and instead gradually leads to their death because the rats are able to detect and warn others to not use the bait. 4. Trap: To better hunt down the rats, it is suggested to set traps at the entrance of active nests after crushing the nests. 5. Motorcycle or any device emitting smoke: One very effective method with the least environmental effects is using smoke from motorcycles. In this method motorcycle exhaust is hosed to the rat’s nest and all holes are blocked so that the smoke chokes the rats (Fig. 10.9). To create more smoke, oil can be mixed in the gasoline of the motorcycle. Instead of a motorcycle, exhaust fumes of sulfur can be used. The best time to fight rats, especially moles, is from mid-March to mid-April. At this time of year, because of animal fertility, they need more food and create the most damage, which leads to easier identification of mole activity at this time of year. As a consequence of feeding on the corms and cutting the stems, the leaves dry up. Dried plants directly show the tunnel created by rats. Smoke or gaseous poisons may then be applied to kill them. 6. Farming operation: As it was mentioned earlier, rat tunnels are often located at farm borders; therefore, plowing borders will destroy nests and rat populations will decrease. Moreover, it is necessary to remove harbors such as debris and bushes to prevent rats from hiding from predators.

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FIGURE 10.9 Controlling rats in saffron fields by smoke. From Rahimi, H., 2016a. Saffron Pests (Survey and Management). Sokhan-Gostar Press, Mashhad, Iran (in Persian).

7. Nontoxic and environmentally friendly components with nanopowders are now on the market. When rats feed on these products, the powder turns into a hard substance that leads to intestinal obstruction and eventually death.

10.4

Pathogens

10.4.1 The fungal agents of saffron corm rot Fungal agents affecting saffron include sclerotineal disease of the saffron corm, which is one of the major fungal pathogens of corm, reported from Jammu and Keshmir in India. The initial signs include small stains on the corm surface that disseminate more each year and become irregularly bordered brown to dark brown stains. These stains deepen to approximately 1mm in the tissue, forming a scar. At this stage, the scar borders are well demarcated from the unaffected tissue. On the upper portions of the aboveground organs, drying and yellowish discoloration appear and extend downward. A white covering is observed on the corms, which are eventually completely decayed (Kalha et al., 2007).

10.4.2 Fusarium rottings of the saffron corm Fusarium solani and Fusarium oxysporum are among the most damaging rotting agents reported in Jammu and Keshmir. (Wani, 2004; Ahmad and Sagar, 2007). The saffron corm infestations are observed as dark brown color, turning to brown sunburned ulcers. These stains have irregular borders and cause rotting mainly near the roots and buds. Eventually the infected corms turn to black powdery masses. On the aboveground organs, the signs include drying and yellowing of the leaf tips (Ghani, 2002). Other fungal agents such as Macrophonia phaseolina (Thakur et al.,1992), Rhizoctonia crocorum, Phoma crocophila (Madan et al., 1967), as well as some saprophytic fungi such as Penicillium hirsurum (green mold) (Fiori, 2002) and Aspergillus spp. (black mold) may cause saffron corm decay. To prevent and control damage by the above agents on saffron corms, 0.2% solutions of tecto (tiabendazole) fungicides or bavistin (carbendazim) fungicides can be used for a 30min immersion followed by drying (Sud et al., 1999).

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10.4.3 Nematodes These are small vermiform organisms that mainly live in soil and cause damage to plants. Some of the parasitic nematodes that cause damage to saffron corms include Helicotylenchus sp., Tylenchorychus, Prathylenchus sp., as well as the free living nematode Xiphinema sp.

10.4.4 The saffron pathogenic bacteria Bacillus croci (Mizusawa, 1923) and Burkholderia (pseudomonas) gladioli pv gladioli (Xu and Ge, 1990) are among the most important saffron damaging bacterial agents. Burkholderia gladioli (Pseudomonas gladioli) is the bacterial agent of soft rotting in saffron new buds and leaves that forms patches on saffron corms and leaves, reducing the flowering rate severely. This disease, reported from Italy (Sardinia), develops during the autumn before flowering, causing decaying of the thin white membrane (a protective layer of the leaves and flowers) of the saffron primary buds (Fiori et al., 2011).

10.4.5 Saffron viruses Viruses are important plant damaging pathogens that mainly cause damage by loss of function. Major identified viruses include: 1. bean yellow mosaic virus (Russo et al., 1979) 2. turnip mosaic virus (Chen, 2000) 3. meadow saffron breaking virus Most of the viruses have vectors like aphids and controlling the aphids could be effective in controlling the agents. Developing virus free plants is one of the most important applications of plant biotechnology, which is the subject of Chapter 14, Tissue and cell culture of saffron, of this book. From the plant pathologists’ point of view, viral disease control could be categorized as: (1) deleting the source of infection, (2) controlling the vectors, (3) direct attack on the virus, (4) producing and breeding resistant variants, and (5) adaptation of specific reproduction methods. Meristem culture is one the most frequent methods for deletion of viruses in infected plants. In this method, meristems from the terminal or lateral buds of the shoots are removed and cultured in meristem specific media. The seedlings are then tested for viral agents. Presence of viral inhibitors in meristem cells, lack of the necessary substances for viral proliferation in meristem cells, lack of developed vascular system in meristems for spreading of viruses, and absence of plasmodesmata in meristem cells for movement of viruses between cells are the most important causes of virus deletion in the method. For more details see Chapter 15, Molecular biology of Crocus sativus. Thermotherapy is the most common virus deletion method. For this, the infected plants are incubated for a specified period of time at high temperature. Then their newly formed buds are cultured, or their meristems are removed. Using heat is an effective way to inactivate viruses. Studies performed on the viruses and their hosts show that when the plants are treated with high temperatures, their viral load decreases. Apparently, the reduction of viruses in the plants undergoing thermotherapy results from the negative effects of heat on viral replication and spreading within the plant. Extended exposure of the whole plant or the cultured tissues to high temperatures usually results in slowing down or inhibition of longitudinal growth or death of the seedlings. Therefore, its efficiency rate is low. In addition, it is mainly limited by the fact that not all viral agents are sensitive to this treatment. Therefore, meristem culturing is used as a complementary method for thermotherapy. Karimi et al. (1993) investigated saffron mycoflora in Iran. In the farm examined, patent symptoms of disease were not observed; however, sampling was carried out on suspected symptoms of the leaves, corms, and roots. The following fungi were isolated and identified: Alternaria alternata, Aspergillus alliaceus, A. niger, A. oryzae, A. terreus, Botrytis sp., Cladosporum herbarum, Embellisia sp., Fusarium equiseti, F. oxysporum, F. solani, F. sp., Phoma eupyrenea, Penicillium spp., Ulocladium atrum. The ecological behavior of each fungus was considered and suitable methods were utilized for investigation of its pathogenicity relative to the saffron plant. For this purpose different species of Fusarium, Botrytis, Phoma, Embellisia, and Penicillium were examined. The only fungi that showed weak chlorosis on saffron leaves was Botrytis. The other species mentioned above did not produce any symptoms on saffron corms, leaves, and roots.

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Conclusion

Keeping in mind the international market value of saffron, it is highly cost beneficial to manage the various pests that reduce quality and quantity of yield in this crop. It should be noted that weeds impose maximum damage to saffron production, insects rank second, and disease has the least impact on saffron. However, few studies have been carried out on harmful biotic factors that adversely affect saffron production. Most methods of saffron farming and pest control are traditional and need to be upgraded with the help of new technologies. Due to climate change, this crop is immigrating to northern latitudes, which may expose saffron to new harmful biotic organisms.

References Abasian, M.R., Bazoobandi, M., Sohani-Darban, A., 2013. Effects of separate and tank mix herbicide application on weeds and saffron corm. J. Weed Ecol. 1 (1), 9
20 (in Persian). Abbaspoor, M., Norouzzadeh, S., Torabi, H., 2011. Investigation of some new herbicides in saffron fields. In: Proceeding of 7th National Horticultural Science, 5
8 September 2011, Isfahan, Iran, p. 574 (in Persian). Ahmad, M., Sagar, V., 2007. Integrated Management of Corm/Tuber Rot of Saffron and Kalazeera, Horticulture Mini Mission-1. Indian Council for Agricultural Research (ICAR), India. Bazoobandi, M., Vojoudi, H., 2016. Saffron Integrated Weed Management. Tohid-Manesh Press, Neyshabour, Iran (in Persian). Behdani, M.A., Jami-Al-Ahmadi, M., Fallahi, H.R., 2016. Biomass partitioning during the lifecycle of saffron (Crocus sativus L.) using regression models. J. Crop Sci. Biotechnol. 19 (1), 71
76. Chen, Ji.S., 2000. Occurrence and control of mosaic disease [turnip mosaic virus] in saffron (Crocus sativus). Zhejiang Nongye Kexue 3, 132
135. Davis, A.S., Schutte, B.J., Iannuzzi, J., Rener, K.A., 2008. Chemical and physical defense of weed seeds in relation to soil seed bank persistence. Weed Sci. 56 (5), (1 September 2008). Eslam-Abbasi, M.A., 1996. Effect of Different Herbicides on Saffron Weed. MSc. Thesis. Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran (in Persian). Farhoudi, R., Rahnama, A., Esmailzadeh, H., 2003. Role of saffron in mix cropping. In: A. Hemmati-Kakhki, M. Allahyari (Eds.), Proceeding of 3rd National Conference on Saffron, 2
3 December 1983, Mashhad, Iran, pp. 173
178 (in Persian). Fiori, M., 2002. Avversita`. In: F. Sanna (Ed.), Zafferano, Storia, Cultura, Coltivazione e Impiego a San Gavino Monreale e in Sardegna. E.R.S.A.T. Centro Zonale di Guspini. Medio Campidano, Sardinia, Italy (in Italian). Fiori, M., Ligios, V., Schiaffino, A., 2011. Identification and characterization of Burkholderia isolates obtained from bacterial rot of saffron (Crocus sativus L.) grown in Italy. Phytopathol. Mediterr. 50 (3), 450
461. Ghani, M.Y., 2002. Corm rot disease of saffron and its management. In: Proceeding of seminar-cum workshop on saffron (Crocus sativus). SKUASTK, India. Kafi, M., Koocheki, A., Rashed-Mohassel, H., Nassiri, M., 2006. Saffron: Production and Processing. Technology. Science Publishers, Enfield, NH. Kalha, C.S., Gupta, V., Gupta, D., 2007. First report of Sclerotial rot of saffron caused by Sclerotinia rolfsii in India. Plant Dis. 91, 1203
1206. Karimi, M.R., Hedjaroud, G., Okhovat, M., 1993. Investigation of Saffron Mycoflora. MSc. Thesis. Tehran University, Karaj, Iran (in Persian). Khoze Mary, A., 2000. Saffron in mix cropping system. J. Saffron 8, 180
244. Knezevic, S.Z., Evans, S.P., Blankenship, E.E., Van Acker, R.C., Lindquist, J.L., 2002. Critical period for weed control: the concept and data analysis. Weed Sci. 50, 773
786. Koocheki, A., Najibnia, S., Lalegani, B., 2009. Evaluation of saffron yield in mixcropping with cereals, puls crops and medicinal plants. Iran. J. Field Crop. Res. 7 (1), 172-163 (in Persian). Koocheki, A.R., Rezvani-Moghaddam, P., Fallahi, H.R., 2016. The study of saffron (Crocus sativus L.) replacement corms growth in response to planting date, irrigation management and companion crops. Saffron Agron. Technol. 4 (1), 3
18 (in Persian). Madan, C.L., Kapoor, B.M., Gupta, U.S., 1967. Saffron. Econ. Bot. 20, 377
385. Makarian, H., Rashed-Mohassel, M.H., Bannayan, M., Nassiri, M., 2008. Spatial dynamics of weed populations in saffron (Crocus sativus) field using Geostatistics. J. Agric. Sci. Natur. Res. 15 (2), 76
85 (in Persian). Manson, D.C.M., 1972. A contribution to the study of the genus Rhizoglyphus robini claparde. 1869. (Acaria: Acarridae) Acorologia. T.XIII. Fasc. 4, 621
650. Marı´, A., 2010. Caracterı´sticas del cultivo de azafra´n (Crocus sativus) en el Valle del Jiloca (Teruel): estudio de la flora arvense y mejora del desherbado meca´nico. In: Proyecto Final de Carrera. Escuela Universitaria Polite´cnica La Almunia de Don˜a Godina, Zaragoza, Spain (in Persian). Mirhashemi, S.M., Bannayan, M., Nezami, A., Nassiri-Mahallati, M., 2015. Evaluation of the extinction coefficient, radiation absorption and use efficiency of saffron (Crocus sativus L.). Saffron Agron. Technol. 3 (3), 203
216 (in Persian). Mizusawa, Y., 1923. A bacterial rot disease of saffron. Ann. Phytopathol. Soc. Jpn 1 (5), 1
12. Mousavi, S.K., Nezam-Abadi, N., Zand, E., Baghestani, M., Shimi, S., 2013. Manual of Weeds Chemical Control in Major Crops of Iran. JihadDaneshgahi Press, Mashad, Iran (in Persian). Naderi-Darbaghshahi, M., Pazoki, A., Banitaba, A., Jalali Zand, A., 2009. Investigation of agricultural and economic aspects of saffron mix cropping with anemis. New Find. Agric. 3, 414
423 (in Persian). Norouzzadeh, S., Abbaspoor, M., Delghandi, M., 2007. Chemical weed control in saffron fields of Iran. Acta Hort. 739, 119
122.

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Nurbakhsh, S., 2016. List of important pests, diseases and weeds of major agricultural crops, pesticides and recommended control methods. Crop Protection Organization of Iran (in Persian). Padarloo, A., 2007. Weed Survey of Saffron Fields of Mashhad. Third International Congress of Saffron, Ghaen, Iran, p. 143. Pir, F.A., Nehvi, F.A., Singh, K.N., Hassan, B., Khanday, B.A., Mir, Z.A., 2008. Saffron weed flora of Kashmir. In: Nehvi, F.A., Wani, S.A. (Eds.), Saffron Production in Jammu and Kashmir. Directorate of Extension Education. SKUAST-K, India, pp. 189
200. Rahimi, H., 2016a. Saffron Pests (Survey and Management). Sokhan-Gostar Press, Mashhad, Iran (in Persian). Rahimi, H., 2016b. An Investigation of Some Physical and Cultural Methods for Controlling Saffron Bulb Mite (Rhizoglyphus robini Claparede 1869). Final Report. Khorasan-Razavi Agricultural and Natural Resources Research and Education Center (in Persian). Rahimi, H., Kamali, K., 1993. Laboratory studies on biology of bulb mite Rhizoglyphus robini (Acari: Acaridae) and its damages on saffron corm in Gonabad and Qaen. Sci. J. Agric. 16, 53
63 (in Persian). Rahimi, H., Mokhtarian, A., Bazoobandi, M., Rahimi, H., Kiani, M., Behdad, M., 2008. Effects of sowing depth and summer irrigation on Rhizoglyphus robini (Acari: Acaridae) population in Gonabad. Entomol. Phytopathol. 85, 1
14 (in Persian). Rahimi, H., Dadmand, M., Torabi, E., Rahimi, H., Torabi, H., Araghi, M., 2013. Investigating the effects of soil solarization on saffron field against bulb mite (Rhizoglyphus robini). Iran. J. Plant Prot. 36 (2), 1
15 (in Persian). Rashed-Mohassel, M.H., Najafi, H., Akbar-zadeh, M.D., 2001. Biology and Control of Weeds. Ferdowsi University of Mashhad Press, Mashhad, Iran (in Persian). Russo, M., Castellano, M.A., Martelli, G.P., 1979. Rhabdovirus-Iike particles in english ivy (Hedera helix) and ivy-leafed geranium (Pelargonium peltatum). J. Phytopathol. 96 (2), 122
131. Salimi, H., Khalghani, J., Gharehdaghi, A.A., Rahimian-Mashhadi, H., 2008. An investigation on weed seed viability in different depths of compost pile. Appl. Entomol. Phytopathol. 76 (1), 103
122. Shabahang, J., Khorramdel, S., Amin-Ghafori, A., Gheshm, R., 2013. Effects on management of crop residues and cover crop planting on density and population of weeds and agronomical characteristics of saffron (Crocus sativus L.). J. Saffron Res. 1 (1), 57
72 (in Persian). Shahrokhi, M.B., Rahimi, H., Rashed, M.H., 2002. Saffron pests, diseases, and weeds. Saffron Production and Processing. Ferdowsi University of Mashhad Publications, Mashhad, Iran, pp. 137
148 (in Persian). Sud, A.K., Paul, Y.S., Thakur, B.R., 1999. Corm rot of saffron and its management. J. Mycol. Plant Pathol. 29, 380
382. Thakur, R.N., Singh, C., Kaul, B.L., 1992. Frist report of corm rot in Crocus sativus L. Indian Phytopathol. 45, 278
282. Wani, A., 2004. Studies on Corm Rot of Saffron (Crocus sativus). PhD Thesis. Shere-e-Kashmir University of Agricultural Science and Technology of Kashmir, India. Xu, C.X., Ge, Q.X., 1990. A preliminary study on corm rot of Crocus sativus L. Acta Agric. Univ. Zhejiangensis 16 (suppl. 2), 241
246. Zare-Hosseini, H., Ghorbani, R., Rashed-Mohassel, M.H., Rahimi, H., 2014. Effects of weed management strategies on weed density and biomass and saffron (Crocus sativus) yield. Saffron Agron. Technol. 2 (1), 45
58 (in Persian).

Further reading Asgarpour, R., Khajeh-Hosseini, M., Khorramdel, S., 2015. Effect of aqueous extract concentrations of saffron organs on germination characteristics and preliminary growth of three weed species. J. Saffron Res. 3 (1), 81
96 (in Persian).

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Chapter 11

Mechanization of saffron production Mohammad-Hossein Saeidirad Department of Agricultural Engineering Research, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran

Chapter Outline 11.1 Introduction 11.1.1 The role of mechanization in agricultural development 11.1.2 Economic advantages of saffron mechanization 11.2 Machines for corm production 11.2.1 Physical properties of saffron corms 11.2.2 Corm digging 11.2.3 Corm sorting 11.3 Tillage 11.3.1 Bed preparation for corm planting 11.3.2 Crust breaking 11.4 Corm planting

11.1

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11.4.1 Planting patterns 11.4.2 Traditional planting methods 11.4.3 Automatic planting machines 11.5 Harvesting saffron flowers 11.5.1 Traditional method of harvesting saffron flowers 11.5.2 Invented picker machines 11.6 Saffron stigma separation 11.6.1 Physical properties of saffron flowers 11.6.2 Traditional stigma
flower separation method 11.6.3 Invented separators 11.7 Conclusion References

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Introduction

11.1.1 The role of mechanization in agricultural development For modern agriculture, it is essential to use implements and machinery. Arguably, these are the most important inputs for the agriculture sector. The goals of mechanization include (Sims and Kienzle, 2006): 1. 2. 3. 4. 5.

increased productivity per unit area due to improved timeliness of farm operations; an expansion of the area under cultivation where land is available; accomplishment of tasks that are difficult to perform without mechanical aids; improvement of the quality of work and products; and a reduction of drudgery in farming activities, thereby making farm work more attractive.

Mechanization systems are categorized as human, animal, and mechanical technologies. Based on the source of power, the technological levels of mechanization have been broadly classified as hand-tool technology, draught animal technology, and mechanical power technology (Sims and Kienzle, 2006). The correct choice of and use of machinery and equipment has a direct effect on yield and income. In general, If development is not restricted in other fields, the use of agricultural machinery can be promoted. Besides the technological development of farm machinery, new technologies should be used in other fields such as seed improvement, irrigation, fertilizers and pesticides, and land consolidation in the agricultural sector. It is only in these cases that agricultural technologies can be used to increase yield, reduce costs, reduce labor, and enhance sustainable agriculture.

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00011-3 © 2020 Elsevier Inc. All rights reserved.

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11.1.2 Economic advantages of saffron mechanization Saffron production is labor-intensive and is the world’s most expensive spice. Saffron cultivation methods have remained almost the same over the last few centuries and thus its production requires high labor hours due to low mechanization of farms (Alonso Diaz-Marta et al., 2006). In addition to the specific climatic requirements for saffron cultivation, labor is another restriction on saffron production in many countries. In fact, 23% of the total energy used to produce saffron is related to manpower. The corm planting, flower picking, and stigma separation are the most important steps in saffron production, and all these steps require a great deal of labor. In a manual harvesting system, 400 and 900 man-hours per hectare are needed for flower picking and stigma separation, respectively (Moayedishahraki et al., 2010). For soil bed preparation before planting, weeding, and crust breaking, equipment available can be used, but planting and harvesting require invention and manufacturing of special equipment and machines. Use of machinery for planting saffron corms can lead to reduced production costs, cultivation of corms in rows and at an appropriate depth, and reduced corm requirement. Mechanization of the saffron harvesting process (flower picking and stigma separation) can reduce microbial contamination of stigmas in addition to lowering production costs. Moreover, yield loss can be avoided due to the shortened harvesting time and reduced need for labor.

11.2

Machines for corm production

11.2.1 Physical properties of saffron corms Saffron corms are spherical with a hard fleshy white texture. Corms are covered with thin brown fibers with thickness increasing toward the outer layers. These thin layers are for corm protection. Saffron corms come in different sizes, and their weight varies from 2 to 40 g (for more information refer to Chapter 7: Saffron corms). Table 11.1 shows the physical properties of saffron corms from different parts of Khorasan province in Iran. Their physical properties including dimensions, geometric diameter, arithmetic diameter, sphericity, particle and bulk density and also static coefficient of friction are given (Bakhtiari-Konari et al., 2013).

11.2.2 Corm digging The flowering life of saffron fields varies from 5 to 10 years depending on the initial corm planting density. By the end of this period, the yield sharply drops due to extensive corm propagation, insufficient space for vegetative growth of the corms, and weak performance of the soil. At this point, saffron corms should be dug out and transferred to a new field. On average, 20
30 tons of corms can be harvested from 5- to 10-year-old fields. Since corms are planted at a relatively large depth (15
20 cm), they are dug out by moldboard plows. Following deep plowing, corms can be separated from soil aggregates by breaking down the latter using hand tools. The mean required tractor working hours and number of workers are 50 hours and 50 persons per hectare, respectively (Saeidirad et al., 2014). TABLE 11.1 Physical properties of saffron corms. Physical properties

Lowest

Weight (g)

Highest

Mean

3.85

14.82

7.77

Geometric diameter (mm)

18.34

27.17

22.96

Arithmetic diameter (mm)

18.42

27.49

23.20

Sphericity (dimensionless)

0.83

0.91

0.86

23

Particle density (g cm )

1.04

1.22

1.19

Bulk density (g cm23)

0.45

0.51

0.48

Static coefficient of friction

0.65

0.73

0.70

Source: From Bakhtiari-Konari, F., Saeidirad, M.H., Garazhian, H., Sahrayei, P., Arianfar, A., 2013. Investigation and comparison some physical properties of saffron corms. J. Res. Innov. Food Sci. Technol. 2, 69
81 (in Persian).

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FIGURE 11.1 Single-bottom moldboard plow for digging out saffron corms.

For example, in most parts of Khorasan province in Iran, a single-bottom moldboard plow is used for this purpose. The tractor passes in a vertical way to the planting direction and makes a 25
30 cm deep furrow. A number of workers follow the tractor and pick up the corms and put them in bags. In this method, the tractor stops at the end of each pass since it moves much faster than the workers, and waits for them to finish picking up the dug-out corms (Fig. 11.1). The low efficiency, high labor requirement, and high labor costs are the main problems of this method. Since the dry conditions of the land at the time of harvest makes it impossible to use tuber harvesters, growers have no choice but to use this method, which is relatively superior to the traditional method that uses shovel and hand tools instead of tractor power.

11.2.3 Corm sorting The harvested corms are attached to each other around the residue of parent corms, which should be separated, and their extra fiber layers must be removed. The corms must then be graded. Since corms weighing less than 6 g are not capable of flowering in the first year, it is thus recommended to separate them from heavier corms. In most regions, saffron corm cleaning and separation are carried out by laborers, which is very time consuming and costly considering the large number of corms produced. Most of the apparatuses designed and developed for corm grading use the cylindrical sieve mechanism, where smaller corms pass through the rotating cylinder bars, and the larger corms roll out of the cylinder along its slope (Fig. 11.2).

11.3

Tillage

11.3.1 Bed preparation for corm planting Bed preparation and planting are important operations, and consume more than 60% of the total energy requirement of the mechanization sector. Therefore, its proper management can play an important role in reducing energy consumption.

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FIGURE 11.2 Schematic view of a saffron corm sorter. 1, Large corms basket; 2, cylinder slope adjustment; 3, small corms basket; 4, feeding conveyor; 5, rotating cylindrical sieve. From Ghanbarian, D., 2012. Design and development of a drum type saffron corm sizing-machine. Agric. Eng. 35(2), 83
96 (in Persian).

Considering the adverse effects of extensive tractor passes on fields during bed preparation, such as soil compaction and erosion, research interest in selection of appropriate implements and increased farm efficiency is growing every day (Tabatabaeefar et al., 2009). Saffron can be planted in most types of soil, whether lightweight sand or heavy clay. However, this plant grows best in medium loamy-texture soils containing humus. Bed preparation for saffron planting requires plowing at a 25
30 cm depth, with the diameter of soil particles ranging from 10 to 15 mm following the tillage practice. This can be done by a chisel plow in mid-April when spring rains are over (Fig. 11.3). Plowing at this time not only is considered as primary tillage but also plays a significant role in weed control. If this is performed at proper moisture content (12%
15%), secondary tillage can be eliminated or at least minimized. It is recommended to spread 20
40 tons of composted manure per hectare before planting and mix it with the soil using secondary tillage implements and machinery such as a cultivator or disc harrow (Behdani and Fallahi, 2016).

11.3.2 Crust breaking Crust breaking is carried out following the first irrigation of the saffron field in the growth season (from late September to mid-November) and when the soil moisture content is 15%
18%. This operation is needed to crush the hardened layer above young saffron sprouts and facilitate their emergence from the soil. Crust breaking in saffron fields during the early growth season must be performed carefully as saffron sprouts have reached near the soil surface (Saeidirad et al., 2007). The tillage depth for crust breaking depends on the distance of the sprouts from the soil surface, which in turn is a function of irrigation time and weather conditions in the region. The later the first irrigation, the closer the saffron

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FIGURE 11.3 Chisel plow equipped with a roller for primary tillage.

sprouts to the soil surface (due to the approaching time of harvest), and thus the crust-breaking operation must be performed with extra care. Growers break crusts using different means and methods such as a tractor-drawn spring-loaded cultivator, rotavator, and powered rotavator. Small tractors (,45 hp) are recommended to be used for drawing cultivators or rotavator. Big and heavy tractors can cause serious damage to crops. Currently, horticultural tractors are common due to their lightweight and high maneuverability in small pieces of land (Sheykhdavodi et al., 2010). A powered cultivator operated by an on-foot user is an example of a machine used for breaking crusts in saffron fields. In this self-propelled tillage implement, the rotavator’s axis where blades are mounted serves as the drive wheel. During road transportation, two wheels replace the rotavator’s two axes. The engine power is transferred through a gearbox to the axes. In crust breaking for saffron, growers have creatively circumvented the possible damage of L-shaped blades to saffron sprouts by replacing them with vertical dual-spike blades (Fig. 11.4). If the first irrigation is performed later than recommended time, which is usually inevitable due to water scarcity, this implement can cause extensive damages to the crops by destroying the saffron sprouts. Therefore, under these circumstances, growers are forced to use traditional methods for crust breaking (Saeidirad et al., 2007).

11.4

Corm planting

11.4.1 Planting patterns Saffron corms can be planted from the end of the growth period in mid-May until early October. It is recommended to plant saffron corms in a new field within a week or two after they are dug out because both air and soil are very warm and the relative humidity of the air is very low causing the corms to incur damage due to moisture loss. Delay in planting corms may lead to emergence of roots in storage and damage during planting, prevented their optimal growth. Digging out corms in warm months (June
August) is not recommended; instead, it should be performed in May or September. Uniform planting of corms in rows can help save more corms. It can also increase yield and facilitate the execution of other field operations, particularly flower harvesting. The planting pattern of saffron in Iran is normally in flat rows. The row spacing varies between 20 and 30 cm. The density of corms per unit area has a direct relationship with its yield and an indirect relationship with the number of the flowing years (age) of the saffron field. As a result, the age of saffron fields ranges between 4 and 10 years depending on the planting density (for more details refer to Chapter 7: Saffron corms).

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FIGURE 11.4 Left, replacement tines for breaking crusts in saffron fields; right, powered tiller rotavator.

It is recommended to mechanize corm planting in order to reduce labor, save the amount of corm required for planting, increase performance speed, and provide agreeable conditions for other mechanized process such as crop protection and harvesting.

11.4.2 Traditional planting methods In traditional planting, growers use single-bottom moldboard plows and tractor-mounted furrow openers to dig furrows with 20
30 cm spacing and a depth of 15
20 cm. A number of skilled workers plant corms in rows with 5
10 cm spacing (Saeidirad et al., 2014). Considering the low row spacing (20 cm) and the fact that corms are planted manually following a tractor pass, two furrow openers cannot be used simultaneously as the soil turned by the furrow openers will pour into the adjacent furrow. This reduces planting speed. In different parts of Khorasan province in Iran, growers have built creative implements and equipment for semimechanization of saffron corm planting. Fig. 11.5 shows a single-bottom moldboard plow equipped with corm hopper. It is drawn by a tractor to open a furrow in the soil. The worker throws corms in a free-fall tube and plant in the freshly opened furrow. The soil from each furrow is then rolled into the previous furrow and thus covers up the corms. The in-row spacing of corms is an effective factor in density and uniformity of the planting operation, which cannot be controlled precisely due to use of labor and its inherent human error. Fig. 11.6 shows a six-row corm planter invented by local farmers used in some parts of Khorasan province. It has a corm box, from which three workers simultaneously pick corms and release them in six free-fall tubes. Its advantage over a one-sided plow is the simultaneous planting of six rows, which increases performance. Similar to the previous method, it requires high labor and is affected by human error in corm spacing.

11.4.3 Automatic planting machines Several implements have been developed for mechanized row planting of saffron corms, most of which use spoon/cup seed metering devices similar to those used in potato planters. Fig. 11.7 is a two-row saffron corm planter capable of planting corms at 20 cm row spacing, up to 5 cm distance between corms. Use of spoon/cup seed metering devices can cause limitations for planting density, and thus are not capable of planting more than 3 tons ha21 of saffron corms. It can plant 0.12 ha h21 with a working speed of 3 km h21 (Saeidirad and Akram, 2006).

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FIGURE 11.5 Single-bottom moldboard plow equipped with corm hopper. From Saeidirad, M.H., Zarifneshat, S., Mahdinia, A., Nazarzadeh, S., Mazhari, M., Mostafavand, H., et al., 2014. Investigation on mechanization development possibility and providing the most optimum method to saffron harvesting mechanization. Final Research Report, No. 44678, Agricultural Engineering Research Institute (in Persian).

FIGURE 11.6 Semiautomated six-row corm planter.

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FIGURE 11.7 The two-row saffron corm planter. From Saeidirad, M.H., Akram, A., 2006. Design and development of two-row saffron bulb planter. AMA-Agr. Mech. Asia Af. 37(2), 48
51.

In Fig. 11.8 a fully automated seven-row corm planter is presented, which was designed and developed at the Khorasan Razavi Agricultural and Natural Resources Research Center, Iran. It is capable of planting 7
10 tons ha21 of saffron corms with 25
30 cm row spacing. Its working width is 210 cm and it can plant 0.8 ha h21 with a working speed of 4 km h21. This seven-row saffron planter consists of a corm container, roller-type seed metering device, furrow openers, corm covering packer, carrying wheels, and power transmission system. This planter can be attached to a tractor by a threepoint hitch, and the seed metering device is driven by the ground wheel. There are two seed metering rollers, driven by the ground wheel, at the bottom of the container that ensures the consistent continuous flow of corms from the container to the openers. Saffron corms are uniformly distributed between the seven openers, and the openers (30 cm apart from each other) are arranged in a nonlinear formation and release the corms at a depth of 20 cm (Saeidirad et al., 2018).

11.5

Harvesting saffron flowers

11.5.1 Traditional method of harvesting saffron flowers Seven to 10 days after the first irrigation (mid-October to mid-November), saffron flowers start to appear on a daily basis. The flowers emerge gradually within a 15
20 day period. The height of the flowers from the soil surface varies from 20 to 120 mm, and reaches 50
160 mm above the petals. Saffron flowers emerge in the early morning hours and grow taller as the day advances and becomes warmer. But the flower height can be affected by other factors such as field age, first irrigation time, and also the quality of the soil and its organic content. The main challenges facing the

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FIGURE 11.8 Fully automated seven-row saffron corm planter. From Saeidirad, M.H., Zarifneshat, S., Nazarzadeh, S., Mehrabi, E., 2018. Design, development and evaluation of 7 rows saffron corm planter. Final Research Report, No. 53999, Agricultural Engineering Research Institute (in Persian).

use of machinery for harvesting are the low height of the flowers from the soil surface and the presence of leaves, which in some cases, particularly when the first irrigation is delayed, emerge simultaneously with the flowers (Saeidirad et al., 2014). In the manual method, the growers harvest flowers in the early morning on a daily basis because the weather is cooler and the flowers are just buds. On the other hand, it is much easier and faster to harvest saffron buds, as in the presence of leaves, it requires more time and effort to harvest the full-blown flowers. Buds are also easier to handle and need less space. However, harvesting buds is more beneficial in that they have longer storage life. Late harvested and full-blown flowers should be rapidly prepared for other operations (separation of stigmas), whereas buds can be storage and wait for 2
4 days under cool conditions and away from sunlight. Buds start to open with sunlight and as the day becomes warmer. As a result, stigmas lose their protective layer of (the petals) and are exposed to sunlight and wind, which may affect quality. Considering the huge workload and insufficient labor of the harvesting process, it is possible to harvest only a part of the flowers as buds (Fig. 11.9). In this case, the buds and flowers should be separated into different containers to help prioritize the next harvest operations in order of storage life. The required labor for harvesting one hectare of saffron depends on the flower density per unit area. Flower density per unit area is itself a function of field age and corm planting density. Flowering is very limited in the first year, and it increases gradually to reach its maximum in the third and fourth years. From this point on, the field yield and flowering rate are reduced. Although the flowering period of a saffron field is 15
20 days long, the peak flowering period lasts no more than 3
5 days; more than 75% of the flowers emerge in this short period and should be picked (Saeidirad et al., 2014) (Table 11.2).

11.5.2 Invented picker machines An important limitation of saffron harvesting machines is the close gap between the flower and soil. Another challenge is the growth of leaves simultaneously with flowers. Emergence of saffron leaves is a function of local weather conditions and also the time of the first irrigation. Delayed growth of leaves after the harvest period is ideal for easy harvesting of saffron flowers. Although the emergence and growth of leaves can be partly delayed by a late first irrigation,

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FIGURE 11.9 Saffron flowers harvesting by hand.

TABLE 11.2 Flowering rate of saffron fields and required labor for flower picking from field. Field age (year)

Saffron stigma (kg ha21)

Saffron flower (kg ha21)

Saffron flower (number m22)

Labor (manhours ha21)

1

2
3

150
250

40
60

75
100

2

6
8

500
700

70
170

180
240

3

8
10

700
800

170
200

240
300

4

9
12

750
1000

185
250

300
400

Source: From Saeidirad, M.H., Zarifneshat, S., Mahdinia, A., Nazarzadeh, S., Mazhari, M., Mostafavand, H., et al., 2014. Investigation on mechanization development possibility and providing the most optimum method to saffron harvesting mechanization. Final Research Report, No. 44678, Agricultural Engineering Research Institute (in Persian).

flowering will also be delayed if the autumn’s cold weather arrives with a delay allowing the leaves to emerge before the flowers. Numerous machines and devices have been designed and developed with this aim. Some the machines are known as hand-supported machines and help workers harvest flowers easier and with less effort. In Spain, in order to make this task easier, a self-propelled machine was developed that allows workers to harvest flowers while sitting (Alonso Diaz-Marta et al., 2006). Also, in Iran, a kind of trolley was introduced to reduce the mechanical damages on the body of labors during saffron harvesting (Fig. 11.10). Another implement known as “Saffron All In One” (SAIO) was also designed and developed that allows four users to perform saffron corm planting, removal of weeds, and harvesting operations while in the prone position (Fig. 11.11). Mechanized flower harvesting is aimed at cutting or picking. Accordingly, several machines have been designed and developed. The cutting mechanism is not applicable when saffron leaves have also emerged with the flowers. This is only useful when leaves grow with delay. The currently available flower harvesting machines can be divided into portable and self-propelled groups. In portable machines, the harvesting head is moved across the field by an operator, and the cut flowers are sucked into a container carried on the shoulders of the operator. Fig. 11.12 shows a portable device for harvesting saffron flowers. The flowers are cut by a cutter operated by the operator and are then transferred into the back-carried container by a suction mechanism. The cutting head consists of two rotary blades rotating in opposite directions. The suction unit has a suction pump powered by a single-cylinder gasoline engine (1 hp). This machine is capable of harvesting 5.5 kg h21 of saffron flowers (Saeidirad et al., 2014).

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FIGURE 11.10 Saffron harvesting with trolley. From Abbaspour-Fard, M.H., Yousefzadeh, H., Azhari, A., Ebrahimi-Nik, M.A., Haddadimoghaddam, M., 2018. Ergonomic evaluation of conventional saffron harvesting versus using a trolley. Saffron Agron. Technol. 6(2), 253
267 (in Persian).

FIGURE 11.11 Saffron All In One (SAIO). From ABAC Holland, 2016. ABAC Holland develops Saffron All-in-One planting and harvesting machine report on prototype II.

Two saffron-picking mechanisms are shown in Figs. 11.13 and 11.14. These devices are still in the research stages and have not been produced commercially. As shown in Fig. 11.13 a prototype based on a cam-strike system was developed for harvesting saffron. This cam-strike system detaches the goblet with no damage to the foils. The cutting process has two stages: First, the two components move toward each other, and second, the flowers and the leaves are caught

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FIGURE 11.12 Portable saffron harvesting device. From Saeidirad, M.H., Zarifneshat, S., Mahdinia, A., Nazarzadeh, S., Mazhari, M., Mostafavand, H., et al., 2014. Investigation on mechanization development possibility and providing the most optimum method to saffron harvesting mechanization. Final Research Report, No. 44678, Agricultural Engineering Research Institute (in Persian).

between these components. Through a linear oscillation, the flower is cut by a series of torsional loads applied to its stem. The cutting process is facilitated by the leaves and their rough surfaces (Gambella et al., 2013). Another prototype uses a two-finger pneumatically powered gripper equipped by a suction (vacuum collection) device. Using the structure of the stem and foils, the gripper detaches the flowers from their stem: it breaks the stem while causing no damage to the detached goblet and the foils. The gripper detaches the flower with its two fingers. It is also made of a body, including the electric motor for powering a fan that sucks in the detached flowers through a vacuum tube, and also a handle equipped with a manually operated pneumatic valve to control the pneumatic gripper. The harvester was experimentally tested on the field for picking saffron flowers in the open fields of San Gavino Monreale, Sardinia, Italy (Manuello Bertetto et al., 2014, 2011).

11.6

Saffron stigma separation

11.6.1 Physical properties of saffron flowers Each saffron flower comprises six petals, three stamens, one triple-filament stigma, and a peduncle. The flowers weigh between 0.35 and 0.45 g with an apparent density of 0.90
0.95 g cm23. Each kilogram of saffron flower contains 2400
2700 flowers. The mechanical and aerodynamic properties of the saffron flower and its parts are given in Table 11.3. The differences in the density and aerodynamic properties of the different parts of the flower allow for solutions for separating them from each other. Most devices and machinery designed and developed for this purpose use these differences.

11.6.2 Traditional stigma
flower separation method The most delicate and time-consuming phase of saffron harvest is the separation of stigma from flowers. The short flowering period in each region makes it impossible to find sufficient labor and time to separate stigma from all harvested flowers on a daily basis. As a result, the growers are forced to store the flowers for a few days. This results in quality degradation and sometime flower spoilage. To achieve high-quality products, the recommendation is to separate stigma no later than 1 day after harvest. The traditional method requires three man-hours for separating the stigmas of 1 kg saffron flower (Saeidirad and Mahdinia, 2014).

FIGURE 11.13 The field operation of the cam-strike harvesting machine: (A) approaching, (B) gripping, and (C) detaching. From Gambella, F., Paschino, F., Manuello Bertetto, A., 2013. Perspectives in the mechanization of saffron (Crocus Sativus L.). Int. J. Mech. Control 14 (2), 3
8.

FIGURE 11.14 The harvesting device. From Manuello Bertetto, A., Ricciu, R., GraziaBadas, M., 2014. A mechanical saffron flower harvesting system. Meccanica 49, 2785
2796.

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TABLE 11.3 Physical and aerodynamic properties of saffron flower parts. Property

Weight (g)

Gravity (g cm23)

Moisture content (%)

Terminal velocity (m s21)

Flower

0.35
0.45

0.90
0.95

88
93

3
3.5

Stigma (three filament)

0.026
0.035

0.90
0.95

72
77

2.8
3.4

Stigma (single filament)

0.008
0.012

0.88
0.94

72
77

3
3.5

Stamen

0.006
0.01

1.10
1.20

84
87

2
2.5

Petal

0.027
0.03

0.82
0.88

88
90

1
1.5

Peduncle

0.1
0.17

0.95
0.99

88
92

3.5
4

Source: Data from Emadi, B., Saeidirad M.H., 2011. Moisture-dependent physical properties of saffron flower. J. Agr. Sci. Tech. 13, 387
398; SanabadiAziz, M., Mostofi, M.R., Faridi, H., 2015. Design and construction of a mechanical machine for separating stigmas from the saffron’s petals. J. Multidiscip. Eng. Sci. Technol. 2(9), 2408
2416; Valeghozhdi, H., Hassanbeygi, S.R., Saeidirad, M.H., Kianmehr, M.H., 2010. Determining coefficient of friction and terminal velocity of saffron flower and its components. Iran. J. Food Sci. Technol. 7(25), 121
133 (in Persian).

FIGURE 11.15 Left, Mancha saffron; right, Bunch saffron.

In Khorasan province, Iran, saffron stigmas are separated through two traditional manual methods resulting in two types of products: Bunch saffron and Mancha saffron (Fig. 11.15). To product of Bunch saffron, the worker cuts open the stem and pulls out the triple-filament stigma along with the white style at the end of it. The stigmas are then placed on one another with their white style. However, to produce Mancha saffron, which is the export-grade product, the flowers are cut from their collar and the triple-filament stigma is then separated from the rest of the flower.

11.6.3 Invented separators As mentioned earlier, cutting should be performed at the peduncle in order to separate the stigma from the flower. In this method, the flower components are dismantled and then the stigma can be separated from the rest of the plant. Therefore, to achieve mechanized separation of stigma from flower, the machine should be able to perform three main steps: flower sorting, cutting, and stigma separation. Fig. 11.16 shows a laboratory-scale sorter for saffron flower developed for a more accurate study of the flowersorting process and possibility of an automated flower-feeding line. It has a cylindrical picker featuring vacuum tines. With every rotation of the cylinder, each vacuum tine picks a flower and releases it on a sloped surface when the vacuum suction is ceased along the way. Due to the asymmetrical center of gravity of the flowers, their peduncle is oriented downward during the free fall. Thus, all flowers are placed individually on the sloped surface and are fed from the peduncle to the conveyor belt (Bakhshi et al., 2018).

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FIGURE 11.16 Saffron flowers sorter. From Bakhshi, H., Abbaspour-Fard, M.H., Saeidirad, M.H., Aghkhani, M.H., Pourbagher, R., 2018. Design construction and evaluation of a row singulator for saffron flowers. Saffron Agron. Technol. 5(4), 361
371 (in Persian).

FIGURE 11.17 Correct cutting point.

Accurate and correct selection of the cutting point is an important technical issue at this stage. Otherwise, loss will increase. The peduncle-petal joint is the best cutting point. By displacing this point, the amount of the white style attached to the triple-filament stigma may increase or decrease (Fig. 11.17). Most researchers use image processing systems to select the cutting point. The change in the color of peduncle from pale purple to deep purple at the cutting point is the main characteristic of automated detection. A schematic view and the operation of the automated cutting device is presented in Fig. 11.18. The device features an automated cutter to separate stigma from the saffron flower and uses image processing to find the right cutting spot. Cup-shaped containers are arranged to collect the flowers and take them to the main system. At the end of the path, the flower is picked up from the cups by two pulleys and is passed through the imaging chamber. The image of each flower is then processed to find the best cutting point. Once the point is determined, the height of the cutting system (at the end of the device) is adjusted according to a computer command, and the flower is finally cut. Following the cutting stage, the vacuum system transfers the stigma and the flower to the storage tank. The results showed the efficiency and high rate of the cutting operation regardless of the shape, size, travel speed, and orientation of the flowers. The efficiency of the device was eight times higher than the manual method (Gracia et al., 2009).

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FIGURE 11.18 Schematic view of the automated cutting machine. C, camera; c, cutting disc; D, air separator; E, transmission gear; f, saffron flowers; M1, M2, M3, M4, motors of the transporter, cutting system, fan and positioning system; P, transmission pulleys; PC, computer; Ps, linear positioning system; Ta, Tm, conveyor belts or flower transporters; Z, area with controlled illumination. From Gracia, L., Perez-Vidal, C., Gracia-Lopez, C., 2009. Automated cutting system to obtain the stigmas of the Saffron flower. Biosyst. Eng. 104(1), 8
17.

FIGURE 11.19 Saffron flower cutting device with manual feeding. From Saeidirad, M.H., Zarifneshat, S., Mahdinia, A., Nazarzadeh, S., Mazhari, M., Mostafavand, H., et al., 2014. Investigation on mechanization development possibility and providing the most optimum method to saffron harvesting mechanization. Final Research Report, No. 44678, Agricultural Engineering Research Institute (in Persian).

A saffron flower cutter developed by an Iranian inventor is shown in Fig. 11.19. Its operation is based on manual feeding by an operator. Flowers are separately fed from their peduncle to the feeding inlet. The flowers pass through two counter-rotating rollers and are cut at the petal
peduncle joint by a vertically moving guillotine-like blade. An important feature of this device is its adjustable blade speed by a central processing system, through which the device can change the cutting point and thus adjust the length of the style attached to the triple-filament stigma. The device features four cutting units and requires three operators. Its capacity with four cutting units is 5 kg h21 of saffron flower, and its mean cutting error, including uncut flowers, is 2.5% (Saeidirad et al., 2014). The same inventor designed and developed a saffron flower separator (Fig. 11.20). The cut pieces (i.e., petals, stigmas, peduncles, stamens) are placed on the conveyor by the operator. The different parts of the flower are separated using airflow, sieve drums, and a magnetic field. It is capable of separating 50 kg h21 of flowers with a separation accuracy of 3% (impurities in cleaned stigmas) (Saeidirad et al., 2014).

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FIGURE 11.20 Saffron flower separator. From Saeidirad, M.H., Zarifneshat, S., Mahdinia, A., Nazarzadeh, S., Mazhari, M., Mostafavand, H., et al., 2014. Investigation on mechanization development possibility and providing the most optimum method to saffron harvesting mechanization. Final Research Report, No. 44678, Agricultural Engineering Research Institute (in Persian).

FIGURE 11.21 Saffron processing device.

The inventor combined the two cutter and separator devices and introduced a saffron processing device in one bundle (Fig. 11.21) made of flower cutter, separator, and stigma dryer units. The dryer is a conveyor type with a netted fabric belt. The heat is supplied by four electric heating elements inside aluminum tubes. The stigma-carrying conveyor travels very slowly on aluminum tubes and the heat from the elements is blown by a fan toward the stigmas and dries them.

11.7

Conclusion

This chapter set out to explore the importance of saffron mechanization, the reasons and motivation for development of machine use in saffron production and the role and impact of intervention on the yield increasing, costs, labor and microbial contamination of stigma reducing. It also sought to show the correct choice and use of machineries and power supplies in different stages of saffron production. Moreover, all implements and equipment currently developed that are available for soil bed preparation, corm digging and sorting, corm planting, crop protection, and saffron harvesting process (flower picking and stigma separation) were introduced.

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References ABAC Holland, 2016. ABAC Holland develops Saffron All-in-One planting and harvesting machine report on prototype II. Available from: ,http:// abacholland.com/wp-content/uploads/2012/09/Report-SAIO-2-1.pdf.. Abbaspour-Fard, M.H., Yousefzadeh, H., Azhari, A., Ebrahimi-Nik, M.A., Haddadimoghaddam, M., 2018. Ergonomic evaluation of conventional saffron harvesting versus using a trolley. Saffron Agron.Technol. 6 (2), 253
267 (in Persian). Alonso Diaz-Marta, G.L., Arghittu, A., Astrka, K., Betza, T., Camba, E., Canadas Sanchez, W., et al., 2006. Saffron in Europe (Problems and Strategies for Improving the Quality and Strengthen Competitiveness). Technical Institute for Agriculture (ITAP), Region of Western Macedonia (RWM), Regional Organisation for Agricultural Development and Technical Support (ERSAT), University of Castilla La Mancha (UCLM), Agricultural University of Athens (AUA). Available from: ,http://www.europeansaffron.eu/archivos/White%20book%20english.pdf.. Bakhshi, H., Abbaspour-Fard, M.H., Saeidirad, M.H., Aghkhani, M.H., Pourbagher, R., 2018. Design construction and evaluation of a row singulator for saffron flowers. Saffron Agron. Technol. 5 (4), 361
371 (in Persian). Bakhtiari-Konari, F., Saeidirad, M.H., Garazhian, H., Sahrayei, P., Arianfar, A., 2013. Investigation and comparison some physical properties of saffron corms. J. Res. Innov. Food Sci. Technol. 2, 69
81 (in Persian). Behdani, M.A., Fallahi, H.R., 2016. Saffron: Technical Knowledge Based on Research Approaches. University of Birjand Press, Birjand, p. 412 (In Persian). Emadi, B., Saeidirad, M.H., 2011. Moisture-dependent physical properties of saffron flower. J. Agr. Sci. Tech. 13, 387
398. Gambella, F., Paschino, F., Manuello Bertetto, A., 2013. Perspectives in the mechanization of saffron (Crocus Sativus L.). Int. J. Mech. Control 14 (2), 3
8. Ghanbarian, D., 2012. Design and development of a drum type saffron corm sizing-machine. Agric. Eng. 35 (2), 83
96 (in Persian). Gracia, L., Perez-Vidal, C., Gracia-Lopez, C., 2009. Automated cutting system to obtain the stigmas of the Saffron flower. Biosyst. Eng. 104 (1), 8
17. Manuello Bertetto, A., Falchi, C., Pinna, R., Ricciu, R., 2011. Laboratory test for an integrated device in agricultural applications. Int. J. Mech. Control 12 (1), 105
112. Manuello Bertetto, A., Ricciu, R., GraziaBadas, M., 2014. A mechanical saffron flower harvesting system. Meccanica 49, 2785
2796. Moayedishahraki, E., Jamialahmadi, M., Behdani, M.A., 2010. Efficiency evaluation of saffron cultivation energy In South Khorasan province. Agroecology 2 (1), 55
62 (in Persian). Saeidirad, M.H., Akram, A., 2006. Design and development of two-row saffron bulb planter. AMA-Agr. Mech. Asia Af. 37 (2), 48
51. Saeidirad, M.H., Mahdinia, A., 2014. Compaction and impaction forces from loading mechanisms for separation of stigmas from dried saffron flowers. J. Agric. Eng. Res. 15 (1), 1
10 (in Persian). Saeidirad, M.H., Mansoorian, N., Behdad, M., 2007. Technical and economical comparision of tillage emplements used for crust breaking of saffron cultivation at different irrigation times. J. Agric. Eng. Res. 8 (2), 93
104 (in Persian). Saeidirad, M.H., Zarifneshat, S., Mahdinia, A., Nazarzadeh, S., Mazhari, M., Mostafavand, H., et al., 2014. Investigation on Mechanization Development Possibility and Providing the Most Optimum Method to Saffron Harvesting Mechanization. Final Research Report, No. 44678. Agricultural Engineering Research Institute (in Persian). Saeidirad, M.H., Zarifneshat, S., Nazarzadeh, S., Mehrabi, E., 2018. Design, Development and Evaluation of 7 Rows Saffron Corm Planter. Final Research Report, No. 53999. Agricultural Engineering Research Institute (in Persian). Sanabadi-Aziz, M., Mostofi, M.R., Faridi, H., 2015. Design and construction of a mechanical machine for separating stigmas from the saffron’s petals. J. Multidiscip. Eng. Sci. Technol. 2 (9), 2408
2416. Sheykhdavodi, M.J., EbrahimiNik, M.A., PourrezaBilondi, M., Bahrami, H., Atashi, M., Seyedian, S.M., 2010. Mechanization planning for tillage of saffron fields using multiple criteria decision-making technique as a policy framework in Iran. Aust. J. Crop Sci. 4 (5), 295
300. Sims, B.G., Kienzle, J., 2006. Farm Power and Mechanization for Small Farms in sub-Saharan Africa. Agricutural and Food Engineering Technical Report (3). FAO, Rome, 2006. Available from: ,http://www.fao.org/3/a-a0651e.pdf.. Tabatabaeefar, A., Emamzadeh, H., Ghasemi-Varnamkhasti, M., Rahimizadeh, R., Karimi, M., 2009. Comparison of energy of tillage systems in wheat production. Energy 34, 41
45. Valeghozhdi, H., Hassanbeygi, S.R., Saeidirad, M.H., Kianmehr, M.H., 2010. Determining coefficient of friction and terminal velocity of saffron flower and its components. Iran. J. Food Sci. Technol. 7 (25), 121
133 (in Persian).

Chapter 12

Emerging innovation in saffron production Mohammad Khajeh-Hosseini and Farnoush Fallahpour Department of Agrotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 12.1 Introduction 12.2 Why is innovation needed? 12.3 Production under controlled environments 12.3.1 Soilless beds 12.3.2 Growth chambers 12.3.3 In vitro cultivation 12.4 Forced flowering 12.5 Nonconventional breeding techniques 12.6 Application of hormones 12.7 Production in inadequate climates

12.1

205 206 207 207 208 208 209 209 209 210

12.8 Organic production 12.9 Mechanization 12.10 Smart farming 12.11 Saffron as feed additive 12.12 Saffron byproducts 12.13 e-commerce 12.14 Conclusion References Further reading

210 210 211 211 212 212 213 213 216

Introduction

Saffron farmers have managed their traditional agroecosystems for centuries based on sustainability of yield with reliance on locally available resources (Koocheki, 1994). Traditional knowledge of farmers is important in saffron production and processing, and there are diverse types of practices that should be recognized, documented, and if necessary modified based on new technologies. However, the importance of economic, social, and cultural values of saffron production as a family farming crop in the area should not be neglected (Koocheki, 2004). Interest in this crop is increasing and it is now being cultivated all around the world from China to Spain and even in Australia and New Zealand since the beginning of the 21st century. Saffron, due to its unique biological, physiological, and agronomic traits, is able to exploit marginal land and to be included in low-input cropping systems, representing an alternative viable crop for sustainable agriculture (Gresta et al., 2008a). In spite of this great potential and the considerable increase in new generation consumer demand for saffron, the future of the plant is still uncertain (Gresta et al., 2008a). Iran is well known as the world’s largest producer and exporter of saffron and produced more than 90% (about 404 tons) of the world’s total annual saffron production from 111,000 ha mostly located in Khorasan Razavi province in 2018 (Donya-e-Eqtesad, 2019). However, the declining trend of the saffron yield since 2000 in the area confirms the necessity for innovation in the production of the crop. Moreover, new initiatives are aimed at strengthening saffron cultivation in nontraditional countries such as New Zealand, the United States, Argentina, and Chile (Fernandez, 2004). The main obstacles to saffron production at a worldwide scale are: (1) lack of access to standard corms and highquality plant material, (2) mismanagement of agricultural inputs, (3) need for genetic improvement and conservation of genetic resources, (4) absence of new production techniques, and (5) loss of technical knowledge and mechanization. The last two are discussed in this chapter.

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00012-5 © 2020 Elsevier Inc. All rights reserved.

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Why is innovation needed?

Until the beginning of the 1980s, Spain almost monopolized the world saffron trade ( . 90%). Its production accounted for 52% of overall global saffron production while India (Kashmir), Greece, Italy, and France produced 21.2%, 13.2%, 7.5%, and 6.1%, respectively (Sampathu et al., 1984). At the end of the 1990s, significant changes in production trends were observed, with Iran taking a lead by annually producing 80 tons of saffron, followed by Kashmir (10 t), Greece (6 t), Spain (3 t), and Morocco (1 t) (Husaini et al., 2010; Saltron et al., 1999). The cultivation area of saffron in Iran has increased and today it is cultivated in 21 provinces (Koocheki et al., 2017). Saffron cultivation plays an important part of the livelihood of family farms. But despite increasing the saffron cultivation area, its average yield per unit of area is decreasing. The yield of saffron depends on many agronomic, biological, and environmental factors and is quite difficult to forecast. Production is influenced by parameters like the storage conditions of corms (Molina et al., 2004c), climatic conditions (Sanaeinejad et al., 2008; Tammaro, 1999, 1990), sowing time (Gresta et al., 2008b), cultural techniques (annual or perennial), crop management (irrigation, fertilization, and weed control), and pests or disease control. Generally, 1 ha of saffron may produce 10
15 kg of dried stigmas, but it can range widely from 2 to 30 kg based on production conditions (Gresta et al., 2008a). There is also a variable range of saffron yield around the world. For instance, the reported yields have ranged from 2.5 kg ha21 in Kashmir, India, and Morocco (Bali and Sagwal, 1987; Sampathu et al., 1984) to 29 kg ha21 in Navelli (Tammaro, 1999). The latter was achieved under irrigated conditions and with using big corms in an annual cropping system. Koocheki et al. (2017) mentioned mismanagement, the loss of technical knowledge and mechanization, lack of access to the standard corms, cultivation under cold and wet environments, and also the droughts and limitations in the irrigation times as the main reasons for saffron yield decline. The limited size of land holdings also makes cultivation less profitable. For example, based on a national report over 75% of saffron farms in Iran are below 1 ha, only 18% of holdings are between 1 and 2 ha, and 7% of holdings are larger than 2 ha (Koocheki et al., 2017). Most saffron-producing regions encounter water-deficit conditions and thus water productivity is another important factor in saffron production. Bazrafshan et al. (2019) estimated the virtual water trade and water footprint of saffron production in different saffron cultivated areas of Iran. They reported that the average water footprint of saffron production in Iran was 4659 m3 kg21. The water footprint of a product can be divided into four components: green, blue, gray (Falkenmark, 1995), and white water (Ababaei and Ramezani-Etedali, 2017). The green water refers to the share of the required water supplied from precipitation. The blue one is related to the amount of the irrigation water applied to produce the product. The gray water footprint is the volume of freshwater required to dilute fertilizers and pesticides used in the production process (Hoekstra and Chapagain, 2008; Hoekstra et al., 2009). The white water footprint is related to the amount of irrigation water lost in a growing season, which is a new concept proposed by Ababaei and RamezaniEtedali (2017). The shares of green, blue, gray, and white water footprints in saffron production were estimated 12%, 42%, 40%, and 6%, respectively (Bazrafshan et al., 2019). The total water footprint of saffron production in Iran was around 1541 million cubic meters (MCM) year21, the share of exported virtual water was 1354.6 MCM year21, and the average economic water footprint of saffron production was 3.1 m3 per US $. Although saffron is one of crops that uses limited water, innovation is still needed to produce higher-efficiency water consumption particularly in arid and semiarid areas that face serious water crises. For instance, the Gonabad region is a part of the central Iranian plateau with an arid and semiarid climate; it has no perennial stream and surface waters and the water resources for agricultural and drinking purposes are limited to seasonal streams and underground waters. This water scarcity poses challenges for agriculture in the area. However, proper use of water resources supplied from the Qanat system has created a unique opportunity for farmers and residents of the region. Promotion of products with a high rate of added value, especially saffron, has had a positive impact on improving the livelihood of residents. Today, saffron cultivation is a profitable job for about 400,000 people in the region. The Qanat system (It is a gently sloping underground channel to transport water from an aquifer or water well to surface for irrigation and drinking.) is a reliable source of water and is based on local and traditional knowledge, which supports preservation of biodiversity and productivity. Traditional saffron cultivation based on Qanat irrigation in Iran has been recognized as one of the Globally Important Agricultural Heritage Systems (GIAHS) by the FAO (FAO, 2018). This system has supplied water for over 2500 years and is still common in some villages. To ensure the future of the saffron crop it is necessary to preserve traditional sustainable methods to improve cultivation techniques, plant materials, and quality evaluation methods and to develop a wide range of saffron uses, particularly those related to human nutrition and health, the subject of many chapters of this book. The worldwide increase in

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the utilization of saffron as a natural product requires new biological and economic development and cooperative programs on technological and medical studies. To fulfill these, however, production systems need to be modernized and high-quality propagation material must be provided to farmers. Technology related to cultivation, postharvest processing, quality control, and product development must also be modernized. To stabilize the production of saffron, new methods and techniques in worldwide marketing need to be developed urgently (Fernandez, 2004). Innovations in saffron production such as cultivation under controlled environments (e.g., under plastic tunnels) (Mollafilabi et al., 2013), in greenhouses (Poggi et al., 2010), the forcing of saffron in cold and incubation chambers (Molina et al., 2004c), and using soilless beds such as hydroponics and aeroponics (Souret and Weathers, 2000) are some of the new growing techniques that can be used to enhance the yield and quality of saffron. Organic saffron production is also in demand internationally to produce healthy saffron and to protect the environment. Smart farming using multidisciplinary and massive data is also a new and powerful tool used to optimize resources.

12.3

Production under controlled environments

The evaluation of land suitability requires a multidimensional approach and its three main criteria for a special crop are physical, economic, and social characteristics, which weigh approximately 48%, 43%, and 9% of the relative importance, respectively, in the land suitability modeling studies carried out by Wali et al. (2016). They evaluated the land suitability of Afghanistan for saffron, and suggested that there are several limitations including environmental and socioeconomic conditions in saffron cultivation in different areas. Cultivation under controlled conditions can be considered as an alternative method of outdoor farming to overcome environmental limitations in inadequate climates (Koocheki and Seyyedi, 2016; Sabet-Teimouri et al., 2010). In some regions, increasing labor costs have made saffron production unprofitable despite its high market price. One way to increase the profitability of saffron production would be cultivation under controlled conditions, where the growth environment and nutrition of the plants can be carefully controlled, resulting in higher yield and generally higher quality (Fallahi et al., 2017; Maggio et al., 2006). This can also be a useful strategy in regions with water-deficit problems (Mollafilabi et al., 2013). Comparably, vertical farming can increase resource-use efficiency, especially water as the main limiting factor for crop production in drylands (Ali-Ahmad et al., 2017). The use of vertical columns, vertically suspended grow bags, and plant factory approaches are some examples of this production system (Touliatos et al., 2016). Vertical production in outdoor environments seems to be mainly appropriate for producing saffron on a household scale to meet family needs. Production under controlled environments can also help growers increase the crop density and extend flowering time. In addition, access to high-quality plant propagations may result in higher yield compared with cultivation under field conditions, which results in higher productivity per land area and per worker.

12.3.1 Soilless beds Accurate regulation of the growth environment (temperature, humidity, and light) and nutrition of plants in hydroponic or aeroponic systems has permitted the growth of plants under conditions in which conventional production is difficult or almost impossible (Souret and Weathers, 2000; Yasmin and Nehvi, 2014).

12.3.1.1 Hydroponics and aeroponics In hydroponics, the plant roots are maintained in either a static, continuously aerated nutrient solution, or a discontinuously flowing nutrient liquid solution (Benton, 1983). In aeroponics, water and minerals are supplied to the plant via a mist that deposits fine droplets of nutrient solution on the roots (Souret and Weathers, 2000). Such soilless culture systems could be implemented as an alternative to the current field culture of saffron (Mollafilabi et al., 2013). The advantages of soilless cultures in saffron production have been shown in several studies. Souret and Weathers (2000) noted that saffron corms grown aeroponically and hydroponically produced more flowers and leaves compared with corms grown in soil. They indicated that aeroponics and hydroponics conditions not only did not reduce saffron stigma production but also significantly increased the dry weight of the corms and the concentrations of crocin and crocetin in the dried stigmas. Maggio et al. (2006) evaluated the adaptability of saffron to soilless cultivation in both a cold glasshouse and a climatic chamber, in which optimal conditions for saffron production were maintained for the entire duration of the experiment. Saffron corm planting density increased and the sprouting and flowering of the corms occurred 10 days earlier in the growth chamber (93 days after transplanting) compared with the glasshouse (103 days after transplanting). The

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yields under control environments also doubled (  22 kg ha21) compared to traditional field cultivation (10 kg ha21). They believe soilless systems represent a valuable alternative to open field saffron production. In addition, the choice of suitable substrates and optimal environmental control possibly increases the flower production and anticipate flower onset (Maggio et al., 2006). There are contrasting research reports about the effect of controlled conditions on the flower yield of saffron in comparison with production under field conditions (Mollafilabi et al., 2013; Sorooshzadeh and Tabibzadeh, 2015), suggesting more research is needed. Several research projects have studied the application of nutritional elements to increase saffron production under soilless cultures. For instance, Sorooshzadeh and Tabibzadeh (2015) evaluated the effect of various copper (Cu) concentrations on the leaves and roots of saffron under hydroponic conditions. Their results indicated that the copper concentration up to 5 μM had a positive effect on both the leaf and root growth of saffron, whereas upper Cu values had an inhibitory effect on saffron growth. One of the other positive aspects of saffron production under control environment, especially soilless cultures, is the possibility of controlling pests and pathogens (Maggio et al., 2006; Renau-Morata et al., 2013).

12.3.2 Growth chambers There are several studies that evaluated the growth, yield, and quality of saffron in growth chambers. Their results are conflicting. For instance, Poggi et al. (2010) indicated that improvement of saffron quality was obtained from incubated corms. In contrast, Garcı´a-Rodrı´guez et al. (2017) showed the possibility of shifting saffron flowering to the offseason by storing corms in ultralow oxygen (ULO) cooling chambers, following by incubation conditions to advance flower initiation and then hydroponic cultivation in a flowering room. Although saffron flowering occurred 2 months later in comparison with corms stored and cultivated under traditional conditions increasing the incubation period significantly decreased the saffron quality based on ISO 3632 standards. Increasing both the ULO and incubation period had negative effects on the total crocetin esters and picrocrocin content, but the safranal content did not show a constant trend. What is certain is that the incubation conditions and the quality of the mother corm have significant effects on saffron yield and quality. Therefore further research is needed to determine the optimum conditions for saffron production in growth chambers.

12.3.3 In vitro cultivation Saffron is a triploid sterile plant (2n 5 3X 5 24) that is vegetatively propagated using daughter corms or cormlets (Fernandez, 2004). Each corm survives for only one season, and each mother corm produces only four to five cormlets in one growing season. Some of the new produced corms, because of their small size or infestation of acari, pests, or fungal pathogens, are not productive in the next growing season. Such low multiplication rates of cormlets compounded in fields limit the availability of adequate planting material and hence drastically reduce productivity of saffron. Therefore corms are indispensable for saffron propagation. Indeed, lack of access to high-quality propagation material with guaranteed levels of purity, homogeneity, and health is one important factor that limits saffron cultivated areas (Renau-Morata et al., 2013). Vegetative propagation of saffron may cause limited genetic variation and genetic erosion of this crop (Ahmad et al., 2011). Thus availability of sufficient quality planting material is another key problem in saffron production (Yasmin et al., 2013). It would take several years to produce the corms required to sow 1 ha (50 corms m22) from an initial corm. Sterility in saffron limits the application of conventional breeding approaches for its genetic improvement, but tissue and cell culture offers great potential for its sustainable production. Conversely, tissue culture methods offer great potential for large-scale multiplication. This fact has led research to be conducted into the in vitro propagation of saffron (Renau-Morata et al., 2013). Studies on the tissue culture of Crocus sativus started in the early 1980s (Parray et al., 2012), and some reports on in vitro cormlet production (Quadri et al., 2010), plantlet regeneration (Majourhat et al., 2007), and somatic embryogenesis (Raja et al., 2007) indicate initiatives in this direction. Yasmin et al. (2013) developed a commercially viable protocol for in vitro corm production in saffron. They used minicorms and corm sections with apical, subapical, and auxiliary meristematic regions as explants. To obtain a source of explants for the in vitro multiplication of saffron that allows low contamination levels and efficient propagation Renau-Morata et al. (2013) produced daughter corms by storing nonplanted mother corms at low temperatures (1 C
3 C) for 9 months. In vitro cultivation of saffron has been briefly explained here and further details are available in Chapter 14, Tissue and cell culture of saffron.

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12.4

209

Forced flowering

Saffron stigmas are typically handpicked from flowers; therefore one of the limitations in saffron production is the labor required for this task. The flowering period of saffron is limited to only 2
3 weeks (Molina et al., 2004a) or 5
6 weeks in some regions, usually during the autumn. Extending the flowering period reduces the intensive labor requirements. Further, it would be much easier to mechanize blossom collection in containerized plants than in those grown in soil (Molina et al., 2004a). Forced flowering is a method of controlling the temperature, humidity, and light conditions to extend the flowering period of saffron. Several studies have published guidelines on forced flowering of corms (Molina et al., 2005, 2004b) and flowering throughout the year by controlling the four phases of saffron growth (e.g., by creating an artificial winter during summer). Molina et al. (2004b, 2005) suggested that the blooming of saffron can be extended by both controlling the temperature and illumination intensity. By corm forcing, flowering time can be spread out in a greenhouse by human control. Therefore blossoming will occur during the whole year and there will not be a labor peak. Although these methods are too expensive and may be unaffordable for producing under industrial scale, they can be considered as innovative methods in the future. Harvest costs depend on the duration of both corm-to-flower and flowering stages, since these may affect the amount and distribution of the hand labor required over time and the regularly scheduled harvest practices. Modulating time and duration of saffron flowering is particularly important to improve yield (Maggio et al., 2006). Under field conditions, flower initiation generally occurs during the summer, simultaneously with the beginning of meristematic activity in the apical bud. Differences in the time required for the flower initiation have mostly been attributed to corm size (Negbi, 1999). In addition, both air and soil temperatures may extend saffron flower induction and flowering duration by up to 2 months (Maggio et al., 2006). Under greenhouse conditions, natural light can be controlled by using automatic blinds and curtains (Tripanagnostopoulos et al., 2004) in combination with elements to decrease the ventilation (Souliotis et al., 2006). Also, in the case of supplementary illumination, it requires additional tools and suitable lights (Wang et al., 2002). This activity is carried out in storage rooms or macrotunnels. Early flowering can be achieved in relation to traditional cultivation in the field by controlling temperature, relative humidity, and lighting through special arrangements and covers.

12.5

Nonconventional breeding techniques

There is a need to increase saffron production and quality to cope with increasing demand. This goal could be achieved, for instance, by obtaining plants with more flowers per plant, flowers with a higher number of stigmas, increasing stigma size, or growing stigmas with increase colorant and aroma, all of which are related to breeding techniques. In plant breeding techniques, traditional methods are based on a massive selection of the best samples among natural or cultivated populations, genetic breeding with wild ancestral species, and spontaneous or induced mutations. The sterility in saffron limits the application of conventional breeding approaches for its further improvement (Fernandez, 2004). There is little knowledge of the genetic background of saffron due to the limited number of researchers working on this topic. Determining saffron genome characteristics can be useful for finding its origin and also for developing more breeding techniques. A number of saffron DNA analyses were carried out by several researchers. In most cases there were not any difference in saffron DNA from different parts of the world, although some phenotypic parameters were different. More details on the related reports to these topics are available on Chapter 13, Utilizing O-mics technologies for saffron valorization, and Chapter 15, Molecular biology of Crocus sativus. The limited genetic variation of this crop, particularly vegetative propagation and the possibility of its genetic erosion, points to the need for further research to determine Crocus biodiversity and to evaluate new breeding techniques for sustainable production of saffron.

12.6

Application of hormones

There are several kinds of hormones and other regulatory chemicals that are used in agriculture to control some aspect of plant development. Phytohormones in saffron have biochemical, physiological, and morphological effects (Amirshekari et al., 2007; Aytekin and Acikgoz, 2008) and can positively influence the development of the root system, the induction of flowering, and the growth stimulation of the plant (Amirian and Kargar, 2016; Azizbekova et al., 1978; Koul and Farooq, 1982). Hormones in saffron production can be applied both during the growing season (Amirian and Kargar, 2016) and on dormant corms (Amirshekari et al., 2007; Greenberg-Kaslasi, 1991) with different purposes; for more details refer to Chapter 7, Saffron corms.

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Production in inadequate climates

Saffron has been adapted to arid and semiarid regions hence its susceptibility to flooding conditions reduces its yield in wet areas. Many of the physiological, morphological, and biochemical processes of saffron can be affected by flooding stress following heavy autumn rainfalls. Increasing ethylene synthesis is a common plant response to various stresses, which can mediate acclimation to flooding stress and the postsubmergence recovery (Sasidharan and Voesenek, 2015). Ethylene biosynthesis is strongly regulated by internal signals and environmental stimuli from biotic and abiotic stresses (Wang et al., 2002). Thus by regulating the production or action of ethylene, the growth and development of saffron can be controlled in stressful environments (Rezvani et al., 2012). Some chemical components like silver ions (AgNO3) have been shown to inhibit ethylene action. Rezvani et al. (2012) evaluated the effect of different concentrations of nanosilver (0, 40, 80, and 120 ppm) on inhibition of ethylene action and the growth of saffron under flooding conditions. Their results showed that soaking saffron corms with 40 and 80 ppm of nanosilver decreased the effect of flooding stress on the root length, root number, and leaves dry weight. More details about the effect of soil and environmental conditions on saffron production can be found in Chapter 6, Saffron water requirements. With regard to climate change influences on the future of saffron cultivation, further research is needed to evaluate the effects of different approaches to reduce the environmental stresses on saffron production.

12.8

Organic production

In most saffron origin areas, evolution of saffron production methods have been based on local conditions, including environmental, social, and agronomical circumstances. In these regions, saffron producers usually do not use agrochemical inputs. They use traditional methods for increasing soil fertility and controlling pests, diseases, and weeds with nonchemical methods based on organic farming principles with minimal impact on the environment, but these practices are not certified. In developing countries, saffron producers are usually smallholders and cannot afford the expensive costs of international inspection and certification. In 1996 the International Federation of Organic Agriculture Movements (IFOAM) proposed an Internal Control System (ICS) based on group certification that is now widely applied to certify smallholder groups in developing countries. ICS is an appropriate system for saffron smallholders because several producers are organized into a single group with an internal support structure and an inspection system in this method (Ghorbani and Koocheki, 2007). Although the concept was historically developed as an exception and solution to address smallholders’ incapacity to access individual certification, there is increasing debate about whether the system is equally or more reliable than individual certification. There are ISO standards for evaluating saffron quality as well as protocols for validating organic saffron. Some countries have even developed their own specifications and quality protocols. For example, the European Union has established rules on organic production, processing, distribution, and labeling. Regulation (EC) No. 834/2007 provides principles, aims, and overarching rules of organic production, and defines how organic products should be labeled (The Council of the European Union, 2007). Some of the key requirements of organic production and processing relevant for saffron based on this regulation are: minimizing the usage of nonrenewable energy and off-farm inputs; recycling wastes and saffron byproducts; using organic corms for cultivation in organic production, cultivation practices and soil management should maintain and increase the long-term soil health, minimizing contamination of the environment, prevention of damage caused by pests, diseases and weeds shall rely primarily on protection by natural enemies, crop varieties, rotation, cultivation techniques and thermal processes, general prohibition of synthetic chemicals, such as synthetic pesticides and fertilizers, prohibition the use of genetically modified organisms (International Trade Centre, 2018). There is rich indigenous knowledge of saffron production around the world that should be documented and integrated with the academic knowledge for successful and sustainable organic production in each region.

12.9

Mechanization

Saffron production is usually based on family farming and by using traditional agricultural practices. There is a lack of improvement in applied traditional cultivation techniques in saffron production. Today, cultivation practices are automated and are performed by different kinds of farm vehicles in a various of crops. For instance, a modified onion planter or a potato planter can be used for saffron corm planting; however, the impossibility of placing the corms with the apex in the upward direction may lead to a delay in emergence and a decrease in the production. There are also

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some specialized tools for saffron cultivation and adapted bulb- and tuber-picking (such as a potato digger) can replace human labor successfully for corm lifting (Gresta et al., 2008a), however, saffron flower harvesting and the separation of the stigma are the most sensitive parts of saffron production and are still performed manually by laborers. The human work required per kilogram of dried saffron required is approximately 400 hours of labor (UNIDO, 2014), out of which 72% goes to flower harvesting and separation of the stigmas. However, the number of workers needed can be reduced in a high-tech greenhouse with a harvesting device. The lack of mechanization of this crop depends is a result of factors like the delicacy of the corms and flowers, which require handling with care, the considerable variation in the corm size, the need to plant regularly, and correctly orienting and placing the corms. Tentative mechanization procedures of some crop techniques for saffron have been carried out (Galigani and Garbati-Pegna, 1999) with limited success. Gathering saffron flowers requires care and intensive manual labor. The flowers only grow a few centimeters above ground and, depending on vegetative activity, might be surrounded by several leaves, which must not be damaged; otherwise, daughter corms will not be produced. The flowers are harvested manually, generally by family members, by cutting the base of the flower stem with the fingernail. Saffron flowers are highly ephemeral and should be picked the same day as the flowering. The best time for picking the flowers is early morning, when the corolla is still closed, and the stigmas have the best quality (Tammaro, 1990; Zanzucchi, 1987). After harvest, stigmas must be separated from the petals and stamen as soon as possible, and is usually done by laborers. Since labor cost is one of the main saffron production costs, mechanization and automation techniques can reduce saffron production costs. Several devices have been proposed to automate saffron production, but most of them do not focus on industrial scales. For more details see Chapter 11, Mechanization of saffron production.

12.10 Smart farming Application of modern information and communication technologies (ICT) in agriculture with the purpose of increasing both quantity and quality of agricultural products is called smart farming. In this system, ICT such as the Internet of Things (IoT), sensors and actuators, geopositioning systems, big data, robotics, precision equipment, etc., are combined with agricultural techniques to create a more productive and sustainable agricultural production process (Kernecker et al., 2018). Smart farming could reduce the negative environmental effects of agricultural practices as well as provide great benefits such as more efficient use of water or optimization of treatments and inputs. In 2011 up to 80% of farmers in the United States used some kind of smart farming technology, while in Europe it was no more than 24% (Lawson et al., 2011). Smart farming should provide farmers with added value in the form of better decision making or more efficient management. Smart farming can be used in saffron production. Its applications are not only suitable for large and conventional farming systems but could also be applied in small-scale farms and complex spaces like family farming systems and organic managed farms (AKIS, 2019). Saffron farmers by precisely measuring variations within a field and adapting their strategies accordingly can greatly increase the effectiveness of pesticides and fertilizers, and by using them more selectively can reduce the negative environmental impacts.

12.11 Saffron as feed additive Traditionally, farmers use saffron waste after harvesting for livestock nutrition. There are several studies on its potential usage as an animal diet and a feed additive in the poultry and livestock industry to promote performance and health. For instance, in the poultry industry, shell eggs naturally are stable against oxidation and can easily be stored under refrigerated conditions, but processed eggs like the dietary modified eggs that contain higher levels of ω-3 fatty acids can be readily oxidized during refrigerated storage (Botsoglou et al., 1998). The use of synthetic antioxidants for increasing the oxidative stability of foods is currently approved, but there is a high demand for natural antioxidants that could replace the synthetic ones and satisfy consumer demands for production of eggs and meat without residues from substances that have the potential to harm human health (Botsoglou et al., 2010). In the last few years, several studies have suggested dietary supplementations like saffron as an effective means for improving the oxidative stability of eggs and meat (Martinez-Tome´ et al., 2001). Saffron byproducts have the same composition as the rest of the spice and can be used as a low-cost sustainable feed additive for its antioxidant and coloring properties, as well as the healthpromoting ones (Botsoglou et al., 2007).

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The major challenges are the standardization of the biological multicomponent composition derived from saffron and the standardization of their effects on animal performance and food quality and safety.

12.12 Saffron byproducts Application of saffron over the years has been limited to food, textile, and pharmacological purposes. After removing the stigmas, the saffron flower is usually considered as waste. Studies indicate the potential of saffron waste for use in cosmetic, fragrance, and flavoring markets. As saffron petals are inexpensive and produced in large amounts they are considered for different purposes. Argento et al. (2010) investigated the composition of the hydroalcoholic extract of saffron dried flowers by using the LC/UV-vis-DAD/ESI-MS (Liquid Chromatography-Uv-visible-Diode Array Detector-Electro Spray Ionization Mass Spectrometry) and GC/MS (Gas Chromatography-Mass Spectrometry) techniques. The GC/MS results indicated a significant compositional similarity between the saffron flower extracts and the commercial cocoa powder aroma. In addition, they found 17 different flavonoids in the LC/MS data, which are well known as antioxidants. Previously, Bergoin et al. (2004) showed a honey note in fresh saffron flowers and suggested its potential use in the flavor and fragrance industry. Honey-like flavor and aroma of saffron were also reported by Lech et al. (2009). On the other hand, saffron petals can be used as an alternative or supplementary medicine in the treatment of some diseases. Hosseini et al. (2018) reported different pharmacological properties of saffron petals such as antibacterial, antispasmodic, immunomodulatory, antitussive, antidepressant, antinociceptive, hepatoprotective, renoprotective, antihypertensive, antidiabetic, and antioxidant in a review paper; most of them are related to the presence of active components in saffron petals that mostly exhibit antioxidant activities. Moreover, Lahmass et al. (2018) indicated the antioxidant properties of ethanolic extracts of six different byproducts of saffron including dry leaves, green leaves, corms, tunics, spaths (part between corm and shoot), and stigmas and suggested that all byproducts produced from the harvesting of saffron stigma could be applied as a natural antioxidant source for biological activities. They found the highest level of free radical-scavenging activity in corms extract and the strangest protection from β-carotene bleaching in spaths extract. Righi et al. (2015) also reported the utilization of saffron petals for phytopharmaceutical and nutraceutical purposes. In another study, Mortazavi et al. (2012) investigated the use of saffron petals in coloring of wool fibers. They found that varied hues from light yellow to light brown were obtained from saffron petals based on the kind of mordants applied and suggested that saffron petal can be a good natural colorant for wool dyeing. Edible extract of saffron petals, particularly anthocyanin, has also attracted the business sector with promising outcomes in Khorasan Razavi province, Iran. Natural edible colorant not only is environmental friendly, but is also believed to have anticancer and antiviral effects as well as other health benefits, as discussed in many chapters of this book. Saffron petals are considered as agricultural waste. Processing and using them can be a new opportunity in the saffron industry, especially considering the huge amount of petals thrown away yearly. For example, around 36 kg of dried petals is produced in each hectare of saffron farm. Considering of 11,000 ha of saffron production area, 3960 tons of dried petals is produced annually in Iran alone.

12.13 e-commerce Electronic commerce (e-commerce) involves using online services including the internet and digital media to sell products or services including agricultural products and services. e-commerce has had a rising trend especially in developed countries hence, many small and medium-sized enterprises in developing countries have the possibility to benefit enormously from e-commerce, which has already resulted in enhanced productivity in a number of areas. One of the leading startups in the agricultural e-commerce field is Keshmoon, which procures and trades saffron (Rhoshanzamir and Roosta, 2019). It was inspired by Persian heritage and traditions, and by using digital technologies has transformed the future of saffron business. Deeply rooted in innovation and integrity, Keshmoon provides new opportunities for growth and profitability and lays a foundation for sustainability. Keshmoon not only introduces sustainable saffron farmers to consumers with an e-commerce platform but also invests a portion of profits in environmental projects with a focus on water conservation initiatives. Farmers, as a result of direct access to consumers, have better profit margins and also pay more attention to environmental conservation, which is important for the sustainability of their agriculture. Keshmoon integrates digital technologies with existing resources in the saffron community into a viable platform that creates and cocreates new forms of growth and value. In each pack you can find a small booklet

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that contains a short history of the farmer, the method of saffron production, and the quality of the product (Rhoshanzamir and Roosta, 2019). It is a story of facts, feelings, and interpretations of saffron growers that transforms the perception of what sustainable cultivation of saffron in Iran should look like. Therefore the goal is not only the business; all of the ethical, social, and environmental aspects of agriculture are important. Wali et al. (2016) modeled land suitability in Afghanistan for saffron production and showed that socioeconomic status is one of the limiting factors in expansion of saffron cultivation. They classified socioeconomic factors into categories such as the motivation of farmers, market channels, revenue costs, availability of labor, road networks, and the marketing information and selling skills of farmers. Introducing successful e-marketing networks and training saffron farmers not only can encourage more farmers to produce this crop, but can increase profits and thus improve socioeconomic status. Processing, packaging, and branding capabilities are important parameters for success of saffron in the world market.

12.14 Conclusion Saffron has been produced in the past, and even in the present, mostly based on family farming systems. Researchers from different disciplines particularly in the food, nutrition, and medicine industries worldwide have started to take interest in this crop and to consider ways to improve farming practices and other aspects of saffron production. The areas of saffron production and trade are expanding to many countries. New technologies were briefly explained in this chapter and some are covered in more details throughout the rest of this book.

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Further reading Ahmad, G.L., Salinas, M.R., Garijo, J., Sanchez-Ferna´ndez, M.A., 2001. Composition of crocins and picrocrocin from Spanish saffron (Crocus sativus L.). J. Food Qual. 24, 219
233.

Section III

Genetics and biotechnology of saffron

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Chapter 13

Utilizing O-mics technologies for saffron valorization Matteo Busconi1, Giovanna Soffritti1 and Jose´-Antonio Ferna´ndez2 1

Department of Sustainable Crop Production, Faculty of Agriculture, Food and Environmental Sciences, Catholic University of the Sacred Heart,

Piacenza, Italy, 2Laboratory of Biotechnology and Natural Resources, IDR/ ETSIAM, University of Castilla
La Mancha, Albacete, Spain

Chapter Outline 13.1 13.2 13.3 13.4

Introduction Analysis of variation in saffron germplasm Epigenetics stability Detection of adulteration and DNA-based traceability

13.1

219 220 222 224

13.5 Perspectives Acknowledgements References

226 227 227

Introduction

Saffron is the spice derived from the dehydrated stigmas of Crocus sativus L. (Ferna´ndez, 2004). C. sativus L. is a species whose botanical origin, despite a very long cultivation history (saffron domestication dates to at least 3500 years ago), is still not clear. Its precious red stigmatic three-branched styles contain both the highly desirable “golden condiment” and a number of secondary chemical derivatives, which have been used in medicine for a number of health properties many of which have been scientifically confirmed or supported in studies (Kyriakoudi et al., 2015). Saffron is renowned as the most expensive spice; its market price ranks among the highest in foods, and it is the highest priced high value agricultural product (HVAP) in the world. To clarify the problem of the botanical origin, a number of DNA analyses have been carried out providing interesting but, unfortunately, not definitive evidence. The karyotype of saffron, whose size has been calculated to be about 10.5 Gb by flow cytometry (Brandizzi and Caiola, 1998), has been analyzed by several authors (Agayev, 2002; Chichiricco`, 1984). The triploid karyotype (2n 5 3x 5 24) is composed of 8 triplets, with triplet 5 containing both metacentric and subacrocentric chromosomes, supporting, along with investigations of nuclear low-copy genes, an allo-triploid origin of saffron. The Crocus cartwrightianus herb is presently recognized as the donor of the diploid genome. Concerning the second possible parent, C. cartwrightianus, C. thomasii, C. pallasii, C. mathewii, C. serotinus, C. hausknechtii, C. michelsonii, and C. almehensis have been mentioned from time to time as possible donors of the aploid genome (Alavi-Kia et al., 2008; Erol et al., 2014; Frello and HeslopHarrison, 2000; Gismondi et al., 2013; Petersen et al., 2008; Tsaftaris et al., 2011). The hybrid origin of C. sativus is also supported by heterosis, expressed in more vigorous perianth parts and style branches compared to those of C. cartwrightianus, and by amphiplasty, wherein a hybrid the formation of a satellite in one of the satellited chromosomes is inhibited by the other(s) (Agayev, 2002). Contrary to this hypothesis, some recent evidence seems to strongly support an autotriploid origin starting from C. cartwrightianus. Nemati et al. (2018), by analyzing multilocus and genome-wide SNPs (single nucleotide polymorphisms), obtained through genotyping-by-sequencing (GBS) clustered C. sativus within C. cartwrightianus samples providing no clear indication that other Crocus species had a contribution to the evolution of the triploid saffron. The high variability present among C. cartwrightianus individuals and the high level of heterozygosity within C. sativus make the autopolyploidization likely, where different C. cartwrightianus genotypes provided to the formation of triploid saffron. In analyzing DNA, much useful information can be obtained: at the genetic level, for example by sequencing and eventually comparing DNAs and sequence variations among different species, or among individuals showing alternative Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00013-7 © 2020 Elsevier Inc. All rights reserved.

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phenotypes within the same species; and at the epigenetic level by studying the epigenetic marks present in the DNAs of different individuals. Epigenetic marks are not associated with nucleotide polymorphisms, and they represent a series of chemical modifications of DNA (DNA methylation) and/or histones (methylation/demethylation; acetylation/ deacetylation) altering strongly the structure of chromatin, influencing, in this way, eukaryotic gene expression and the development and environmental responses of plants likewise. Presently, most plant epigenetics studies have been mainly focused on DNA methylation due to the presence of mechanisms of inheritance and because of the relative simplicity of investigating it at a genome-wide level (Seymour and Becker, 2017). The most frequent mark of DNA methylation is represented by cytosine methylation-originating 5-methylcytosine (5-mC), which in plants can be present at different sites having CG, CHG, and CHH (H represents any base but G) sequences (Wischnitzki et al., 2015). Contrary to DNA sequence, epigenetics marks are strongly influenced, among other factors, by the life or seasonal cycle, different tissues or organs, environment, and the interaction among organisms and the environment itself. Other than supporting the study of the history and evolution of the species, DNA analysis can be successfully applied to solve other important topics such as germplasm characterization and analysis of genetic diversity present within the species, identification of useful traits for crop improvement, and development of traceability methods to detect and prevent adulterations and frauds. Some of these aspects will be better explained in the following paragraphs.

13.2

Analysis of variation in saffron germplasm

The loss of land surface cultivated with saffron crop in many areas, mainly in Europe, has very likely led to a strong genetic erosion. Hence, the creation of a germplasm bank for this species, including wild relatives to broaden the gene pool available for genetic improvement, represented a great achievement. Since 2007, the “CROCUSBANK” project (European Commission AGRI GEN RES 018 Action: Genetic Resources of Saffron and Allies—Crocus spp.) and subsequently a number of Spanish projects, have permitted the creation, management, and implementation of the World Saffron and Crocus Collection (WSCC) located in the Bank of Plant Germplasm of Cuenca (BGV-CU), Spain (see European Commission, 2013). The germplasm contains a reasonably good representation of saffron Crocus and allied biodiversity, although there is an abundance of Spanish accessions (see http://www.crocusbank.org for details). The germplasm bank includes almost 600 accessions representing 70 different Crocus taxa, with more than 240 C. sativus accessions from at least 15 countries that represent the largest world-scale collection of saffron Crocus genetic resources (Table 13.1). Germplasms, with the constitution of catalog fields, represent one of the most spread forms of ex situ conservation strategies to preserve genetic diversity. The management of a germplasm is not an easy task for different reasons (lack of funding, personnel, infrastructure), and consequently, the efforts required to preserve this big number of accessions can be justified only in the presence of genetic variability and, in order to detect variability, DNA analyses are mandatory. While genetic differences among Crocus species are evident, the situation is not as simple for C. sativus. Actually, because of its genetic triploid constitution, saffron is sterile and, since the first cultivations, has been obligatorily propagated vegetatively, year by year, via the production of new corms replacing the old ones (Ferna´ndez, 2004). Corm multiplication does not generate genetic variations with the exception of some spontaneous mutations and, TABLE 13.1 List of the accessions of saffron and other Crocuses preserved and propagated at the WSCC germplasm. Accessions

Autochthonous materials collected in Spain (with original passport data)

Autochthonous materials collected in other countries (with original passport data)

Materials obtained from specialised nurseries and other sources (without original passport data)

Total accessions

Saffron (Crocus sativus L.)

158

77 (12 countries)

8

243

Other Crocuses (near 70 species and gardening hybrids)

128

64 (6 countries)

158

350

Total

286

141

166

593

The different numbers have been communicated by Dr. Marcelino De-Los-Mozos Pascual, Curator of the WSCC.

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consequently, the presence of any genetic variability has been debated for years. Despite an extremely high economic value, saffron is presently considered as a neglected and minor crop and research is very far behind research on other crops or model plants and the amount of information available, concerning important aspects such as genetics and epigenetics, is very limited. In a preliminary characterization of 50 saffron accessions of the WSCC, characters related to phenology, floral morphology, and saffron production were measured highlighting a large variability (De-Los-Mozos-Pascual et al., 2010; Ferna´ndez et al., 2011). Similarly, phenotypic variations have been frequently observed in the field by researchers and saffron growers: plants with a different number of stigmas or with a different aspect of tepals. Interestingly, such phenotypic variations are occasionally unstable and can change from one growing season to another (Ferna´ndez, 2004). The biggest evidence, up to now, supports a very low level of genetic variability among saffron accessions as detected by using molecular markers such as simple sequence repeats (SSRs), EST-derived SSRs, and amplified fragment length polymorphisms (AFLPs) (Fluch et al., 2009; Siracusa et al., 2013). Single nucleotide polymorphisms (SNPs) in different accessions, which are often heterozygous, have been detected by direct sequencing (Ferna´ndez, unpublished data). Lately, as a result of the biggest study carried out up to now to clarify the presence and extent of genetic variability in saffron germplasm, 112 accessions of the WSCC with different geographic origin were analyzed. We detected clear evidence about the presence of a small but significant genetic variability, likely consequently the prolonged vegetative propagation, to end the debate concerning the presence, or absence, of genetic variability among saffron accessions (Busconi et al., 2015). Two main clusters were defined by AFLP markers—the first one including only Spanish accessions and the second one including both Spanish and non-Spanish accessions. At the moment, no evidence is available concerning a possible link between this genetic variability and the high frequency of alternative phenotypes frequently observed in the field. However, Agayev et al. (2009) were able to find fast and stable responses of saffron accessions to clonal selection. By applying clonal selection, the authors were able to select population of corms characterized by very different phenotypes. The high phenotypic variability, in the absence of a significant genetic variation, supported the presence of something different from solely genetics in determining these different phenotypes, raising the question about the possible origin of these alternative phenotypes. Considering that gene expression can be influenced by both genetic and epigenetic changes, epigenetics could be a possible origin of the frequently observed alternative phenotypes. In the study previously reported (Busconi et al., 2015) the same WSCC 112 accessions stored in the germplasm were analyzed not only at the genetic level by AFLP markers, but also at the epigenetic level by using methylationsensitive AFLP (MS-AFLP) markers. The results were surprising. In fact, the percentages of polymorphic peaks were 4.23 and 33.57 by using twelve AFLP and three MS-AFLP selective primer combinations respectively. This evidence shows a very high epigenetic variability with respect to the genetic one that could be a possible explanation of the alternative phenotypes. Epigenetic variations are strongly influenced by environmental conditions. In the reported study, we considered accessions with different geographic origin, so in order to reduce the effect of the origin as much as possible, samples were grown under open field conditions in the same field for at least 3 consecutive years to obtain better standardization of the epigenomes. In fact, although epigenetic states are stable by definition, they can revert at certain frequencies (Paszkowski and Grossniklaus, 2011). Despite this, epigenetic variation was still very high and enough to make possible the separation samples according to the geographic origin. As evident from Fig. 13.1 Spanish accessions from the WEST of Central Spain: provinces of Toledo and Ciudad Real belonging to Castilla
La Mancha autonomous community (a.c.), and from the EAST: provinces of Cuenca (Castilla
La Mancha a.c.) and Teruel (Aragon a.c.) were clearly different at the epigenetic level. The same separation was not possible by considering genetic variation. Climatic and edaphic conditions between the two areas are very different and this may be reflected in the epigenetic composition. These results clearly show that epigenetic variability is definitely higher than genetic variability among saffron accessions with different geographic origin. Epigenetic variability can be a consequence of the original different growing areas possibly influencing and determining the alternative phenotypes detected in the field. Thus epigenetics can be considered to differentiate accessions based on the area of origin. As for genetics, also for epigenetic variability, presently, there are no evident links between epigenotypes and particular phenotypes even if some possible evidence can be obtained from a recent study (not published). In the work carried out by our research group and presently under submission, five accessions characterized by stable different phenotypes (pigmentation of tepals, high and low saffron production, early and late flowering time) and low genetic variability, as detected by AFLP markers, were analyzed by applying a methylation-sensitive restriction enzyme-sequencing approach (MRE-seq) coupled with high-throughput sequencing. Interesting results were obtained, in particular: (1) many differentially methylated regions were detected, confirming the high epigenetic variability present among saffron accessions, including sequences encoding for proteins that could be good candidates to explain the accessions alternative phenotypes. In particular, transcription factors important for the flowering process (MADS-box and TFL) and for the production of

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FIGURE 13.1 Factorial correspondence analysis showing multivariate relationships among MS-AFLP epigenotypes of Spanish accessions. WSCC accessions from the WEST (Toledo and Ciudad Real, on the left, dark gray) and from the EAST (Cuenca and Teruel, on the right, light gray) tended to cluster separately with just few exceptions. Modified from Busconi, M., Colli, L., Sa´nchez, R.A., Santaella, M., De-Los-Mozos Pascual, M., Santana, O., et al., 2015. AFLP and MS-AFLP analysis of the variation within saffron Crocus (Crocus sativus L.) germplasm. PLoS One 10 (4), e0123434.

pigments (MYB) were detected; (2) a high number of SNPs, likely arisen in consequence of the prolonged vegetative propagation, were found evidencing a surprisingly high genetic variability, contrary to what was detected by AFLP. This difference can be based on the different techniques used and that, in vegetatively propagated species, next generation sequencing (NGS) approaches are definitely better in order to detect the presence of genetic variability. The conclusion from these experiments is that saffron is not a monomorphic species. Different accessions can be characterized by genetic and epigenetic variability and this justifies the efforts to preserve genetic resources in a dedicated germplasm.

13.3

Epigenetics stability

Because of its peculiar triploid genetic constitution, saffron is a sterile species that can be propagated only in a vegetative way. This way of propagation prevents crop improvement, for phenotypes of interest, through classical breeding approaches such as mating of individuals with the desired characters and selection in the progeny of the plants with the desired traits. Despite this, crop improvement in saffron is possible as demonstrated by Agayev et al. (2009) evidencing a rapid and stable response of saffron to clonal selection. Different economically important species for agriculture are presently propagated in a vegetative way in order to propagate and preserve ideal varieties. Several fruit crops are the result of prolonged clonal propagation, through grafting or in vitro culture, of single founder plants. Several studies carried out in recent years have shown that a continuous vegetative propagation could result in the appearance of

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stable epigenetic variants that can influence, for better or for worse, the phenotype of the regenerated plants. Ong-Abdullah et al. (2015) discovered that a differentially methylated region is responsible for the formation of the mantled trait in trees propagated through tissue culture. The mantled trait is an aberrant phenotype that destroys the production of the trees. In grapevine, the difference between red and white grapevine varieties is due to mutations in two genes encoding for MYB transcription factors. Interestingly, in white cultivars, the expression of one of the two genes, VvMYBA1, is blocked because of the insertion of a retrotransposon in the promoter, leading to epigenetic changes resulting in the inactivation of the gene (Walker et al., 2007). The presence of epigenetic variants consequently continuous in vitro culture has been detected in many plant species, such as grapevine (Schellenbaum et al., 2008), agave (Dı´az-Martı´nez et al., 2012), and coffee (Bobadilla Landey et al., 2013). For vegetatively propagated crops, epigenetic variations and epigenetic-based phenotypes could represent an added value for crop improvement, but in order to use epigenetic information it is fundamental to better understand the stability of epigenomic patterns in organisms (Springer and Schmitz, 2017), especially under natural conditions. In fact, several plants are vegetatively propagated by in vitro culture and using greenhouses or growth chambers; contrary to this, propagation in the field is the norm for saffron. If epigenetic patterns can be stable through development and consecutive years, then the epigenome could describe the epigenetic profile of an organism and to predict traits. Recently, in order to deepen existing knowledge on saffron epigenetics and to define epigenetic stability in consecutive years of vegetative cultivation in the field, 17 accessions, selected because of their different geographic origin, were analyzed (Busconi et al., 2018). Before the analyses all the accessions were cultivated for at least 3 consecutive years in the same field to reduce, as much as possible, the effect of the origin. Accessions received in the saffron germplasm collection in various years, ranging from 2005 to 2010, were considered to highlight whether the epigenotypes may somehow be influenced by the time of cocultivation in the same field. All the accessions were cultivated in close proximity, in order to exclude possible effects on the epigenetic profile of the samples as a consequence of possible different soil composition. For each accession, samples were collected in four consecutive growing seasons, 2012
13, 2013
14, 2014
15, and 2015
16, at the end of the vegetative period. The analysis was carried out by using a MSAFLP approach by using, as methylation-sensitive enzymes, two isoschizomers (MspI and HpaII). These enzymes are able to cut the corresponding cutting site (50 CCGG30 ) in the absence of cytosine methylation, while they cut in a different way in the presence of cytosine methylation. Some of the results of the analyses are given in Fig. 13.2.

FIGURE 13.2 Results of the principal coordinate analysis (PCA) of the epigenetic variation, among and within accessions, evidenced with the MSAFLP analysis. The restriction was performed using, as methylation-sensitive enzyme, HpaII. Different accessions are colored based on the year they were received at the germplasm. It is evident that the different accessions, despite a small level of intraaccession variability, are characterized by different epigenotypes. Each accession is labeled with its corresponding number of the WSCC (e.g., BCU001619). Accessions BCU001584, 1619, 1649, 1672, 1687, 1698 were received in 2005
2006. Accessions BCU001747, 1754, 1806, 1697, 1782, 1783 were received in 2007. Accessions BCU002476, 2874, 2930, 2479, 2708 were received between 2008 and 2010. Modified from Busconi, M., Soffritti, G., Stagnati, L., Marocco, A., Marcos Martı´nez, J., De-Los-Mozos Pascual, M., et al., 2018. Epigenetic stability in saffron (Crocus sativus L.) accessions during four consecutive years of cultivation and vegetative propagation under open field conditions. Plant Sci. 277, 1
10.

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TABLE 13.2 Analysis of molecular variance (AMOVA) for the two methylation-sensitive enzymes (MspI and HpaII) showing the impact of variability among and within accessions in explaining the epigenetic variability and the changes observed during the years of the experiment. Enzyme

Source of variation

Degrees of freedom

Variance component

Partition percentage

MspI

Among accession

16

17.823

96

Within accession

51

0.662

4

Total

67

18.484

100

Among accession

16

21.021

92

Within accession

51

1.882

8

Total

67

22.903

100

HpaII

Source: Modified from Busconi, M., Soffritti, G., Stagnati, L., Marocco, A., Marcos Martı´nez, J., De-Los-Mozos Pascual, M., et al., 2018. Epigenetic stability in saffron (Crocus sativus L.) accessions during four consecutive years of cultivation and vegetative propagation under open field conditions. Plant Sci. 277, 1
10.

The analyses evidenced an important aspect: the different accessions were characterized by highly different epigenotypes. Epigenetic variations are influenced by environmental conditions, so it is not to be excluded that samples from growing areas under different climates could be characterized by different epigenomes. To reduce this effect all the accessions, after being received at the saffron germplasm, were cultivated in the same small field, under open field conditions, for at least 3 consecutive years trying to decrease, as much as possible, epigenetic variations between accessions before the analysis (some epigenetic states can revert at certain frequencies) (Paszkowski and Grossniklaus, 2011). After the analysis it was possible to observe that, independently by the cocultivation before the study and by the continuous cocultivation along the 4 years of the study, all the accessions had at the beginning and maintained, with just minor changes, a characteristic epigenotype different from that of the other accessions (Fig. 13.2), supporting a strong stability of the epigenetic structure in saffron. It is possible to hypothesize that the obligatory vegetative reproduction of saffron can play an important role in maintaining stable, in consecutive years, the big part of the epigenotype independently by the environmental conditions. According to some authors, the reversible changes, despite the epigenetic base, should be considered as part of the phenotypic plasticity while a true epigenetic mark should be stable and stably transmitted to the progeny through cellular divisions (meiosis and mitosis) for several cycles (Richards, 2011). At the end of the experiment it was possible to state that all the accessions maintained a typical epigenotype and, concerning intraaccession variability, it was possible to note that, during the 4 years of the project, all the considered accessions underwent a small number of epigenetic changes slightly modifying the epigenotype of each accession. The analysis of the molecular variance (Table 13.2) evidenced that intraaccession variability is smaller than the among accession variability, explaining a percentage of the 4%, MspI, and 8%, HpaII of the total variance (Busconi et al., 2018). In saffron, it is frequently reported that alternative phenotypes can be both stable or unstable in subsequent growing seasons. Based on the results we obtained, stable and unstable phenotypes could be a consequence of, respectively, stable and unstable epigenetic marks. As reported by Springer and Schmitz (2017), if the inheritance of DNA methylation is stable, any epiallele will be faithfully inherited. In contrast, if DNA methylation patterns are unstable, then the rapid formation, or loss, of epialleles within populations is expected. In the first case, for saffron, epigenetics could be important for breeding purposes while in the second case this is not so.

13.4

Detection of adulteration and DNA-based traceability

Saffron can commercialized in the form of entire dried stigmas or as a finely ground powder. Among the major candidates for adulteration, saffron is one of the most targeted foods and spices and adulterations are a real concern for the saffron market. Adulterations are more often performed in ground stigmas, where extraneous material can be more easily mixed (Torelli et al., 2014). Over the years, adulterations have been detected involving the addition of plant species, animal-derived substances, and synthetic dyes, among others (Europe saffron White Book. Available at http://www. europeanSaffron.eu/archivos/White%20book%20english). Among the most-frequent plant materials used to adulterate saffron, it is possible to cite: (1) cut and/or dyed C. sativus stamens; (2) petals of safflower and calendula

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(Carthamus tinctorius L. and Calendula officinalis L.); (3) powdered rhizomes of curcuma (Curcuma longa L.); (4) fruit extracts from Gardenia jasminoides Ellis; and (5) flower dyes extracted from flowers of Buddleja officinalis Maxim. In addition, commercial safflower and curcuma are often mislabeled under the name “saffron” to mislead consumers. Presently, the biggest number of studies, to detect adulteration and false declarations of origins, have been carried out using chemical and physical approaches among which are chromatographic-based methods and nuclear magnetic resonance spectroscopy (Petrakis et al., 2015). Recently, modern genetic fingerprinting techniques based on the analysis of DNA have been proposed and applied. They represent alternative low-cost technologies enabling once-adequate methods to recover and to amplify DNA from the different matrices are available the identification of individual plant material in raw matrices and in processed food. DNA based methods are highly specific requiring very small amounts of sample for analysis, potentially even micrograms, and they are the only possibility to provide, without uncertainty, the exact name of the adulterant species that has been added. Recent papers have dealt with the development of molecular markers for saffron traceability (Babaei et al., 2014; Marieschi et al., 2012; Torelli et al., 2014) and with alternative methods based on barcoding melting curve analyses using the universal chloroplast plant DNA barcoding region trnHpsbA (Jiang et al., 2014). In a paper of our group (Soffritti et al., 2016) carried out in the frame of the European Cooperation in Science and Technology COST Action FA1101 (Omics Technologies for Crop Improvement, Traceability, Determination of Authenticity, Adulteration and Origin in Saffron: SAFFRONOMICS), we were focused on two main problems: (1) adulteration with different plant species (curcuma, gardenia, safflower, buddleja, and calendula) and (2) adulteration with different parts of the saffron flower. While the first point can be addressed by developing DNA-based markers specific for the different species, the situation is different in the second case, since the DNA of the different parts of the saffron flower are the same. Concerning the first point, an example of the efficacy of the developed markers in evidencing the presence of adulterant species is shown in Fig. 13.3 relatively to the case of saffron and curcuma. To validate markers, artificially adulterated samples were prepared, before the DNA extraction, by mixing two species (saffron and the adulterant) in different percentages from 50%/50% to 99.5%/0.5%. It is important, in order to simulate the real situation that can be present on the market, to mix the species before the DNA extraction and not to mix the different DNAs after the extraction has been performed. As evident from Fig. 13.3 saffron and curcuma specific markers are able to amplify, as expected, just in the corresponding species and in the three mixtures (from 80% saffron/20% curcuma to 95% saffron/ 5% curcuma). Interestingly, the curcuma specific marker was able to detect the presence of the adulterant DNA even at the lowest percentage (Fig. 13.4). Using species markers, we were also able to detect the adulterant species in extremely low percentages (99.5% saffron/0.5% adulterant species). According to the ISO 3632 for saffron, for saffron belonging to categories 1 and 2, the level of unwanted contaminations with material from other plants is 0.1% and 0.5%, respectively. DNA methods are sensible enough to detect these low levels of adulterant. We think that because of this high sensitivity, DNA-based techniques must be applied with different steps: (1) qualitative PCR to confirm the presence of extraneous plant material and (2) in positive samples, trying to quantify the relative amount of extraneous DNA by quantitative approaches. Concerning the detection of self-adulteration by using DNA analyses, the situation is different. Saffron can be considered as one of the main adulterants of saffron itself. In particular, the addition of tepals and/or stamens, cut in pieces and colored, is one of the most widespread adulterations carried out mainly in the powder form. While the identification

FIGURE 13.3 PCR amplification carried out using saffron and curcuma markers on artificial mixtures. (A) Amplification performed using the Crocus-specific marker and (B) amplification performed using the curcuma-specific marker. As expected, the amplification is present just in Crocus, curcuma, and in the mixtures. The ratios refer to the different amounts of saffron and curcuma powders that were mixed before the DNA extraction. Modified from Soffritti, G., Busconi, M., Sa´nchez, R.A., Thiercelin, J.M., Polissiou, M., Rolda´n, M., et al., 2016. Genetic and epigenetic approaches for the possible detection of adulteration and auto-adulteration in saffron (Crocus sativus L.) spice. Molecules 21 (3), 343.

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FIGURE 13.4 Epigenetic profiles obtained by using the MS-AFLP markers on DNA from different parts of the Crocus flower. The analysis was carried out by using an ABI3100 genetic analyzer (Applied Biosystems). The different peaks correspond to DNA fragments of different size. It is immediately visible that the epigenetic profile of the whole stamen is very different with respect to the epigenetic profile of filaments and other parts of the flower that are more conserved. Other polymorphisms are also evident among stamens and the other parts of the flower. Modified from Soffritti, G., Busconi, M., Sa´nchez, R.A., Thiercelin, J.M., Polissiou, M., Rolda´n, M., et al., 2016. Genetic and epigenetic approaches for the possible detection of adulteration and auto-adulteration in saffron (Crocus sativus L.) spice. Molecules 21 (3), 343.

of different species can be obtained by developing specific markers, the situation is different when the adulterant is part of the same plant because the genetic profile is basically the same. In addition, in saffron, because of a little genetic diversity (Alsayied et al., 2015; Siracusa et al., 2013) no different characterized cultivars are present as in other crops. Because of this, detecting adulteration with other parts of the same flower is not as simple to achieve by using molecular approaches. Epigenetics can offer a possible solution. Epigenetic changes, other than the geographic origin, can also be associated with the different parts of the same organism. Starting from this, pools of tepals, stamens, and stigmas collected at the WSCC of Cuenca were analyzed at the genetic and epigenetic level by using AFLP and MS-AFLP approaches. For each part of the flower, different pools were considered. As expected, AFLP analysis provided only a very small amount of possible polymorphic and informative DNA fragments; on the contrary, the methylation-sensitive analysis provided results that are more interesting. It was immediately evident that the epigenetic profile of the whole stamen (first panel in Fig. 13.4) is clearly different from the profiles of the other parts. Stamens are the pollen-producing organ of the flower; it is divided into filament and anther-producing pollen granules. Whole stamen epigenetic profiles were significantly different, presenting a high number of polymorphisms, both among the different parts of the flower and among the different pools of stamens analyzed (red and blue signal in the first panel of Fig. 13.4). Removing the anthers, the epigenetic profile of the only filament was more similar to the profiles of the other parts. Very likely, the observed polymorphisms were a consequence of the anthers and, likely, of the sterile pollen granules present inside them. Excluding the anthers, the epigenetic profiles of the different parts of saffron flowers were more similar but a higher number of polymorphic signals were detected with respect to the variability detected with the corresponding genetic profiles. This finding open to the possibility to develop a traceability method based not on genetic but on epigenetic analysis. Other studies will be required to develop epigenetic markers that could be used as traceability markers for detecting both self-adulteration and saffron coming from different geographic origins.

13.5

Perspectives

Despite several positive aspects, an extremely high economic value, a high sustainability of cultivation, and a high linkage with our cultural and agricultural heritage, saffron is still considered as a largely neglected and minor crop, seen as having nothing or little to offer to modern agriculture, which is currently dominated by just a handful of crops. Because of this, saffron has frequently given way to major crops, such as grains, and this situation has been observed both in the field and in research. In the field, with the strong genetic erosion and loss of a vast agricultural surface previously cultivated with saffron; in research, with very few research projects being supported by the authorities in recent years. Consequently, saffron research is presently very far behind research other crops and the amount of information available is limited. To be fully exploited in the field and thus render saffron a remunerative crop for both farmers and the

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environment, a lot of research must be undertaken. Knowledge on saffron must clearly be improved and, as for many other crops, the further step to achieve this goal is the application of omics methodologies. Concerning nucleic acids, these approaches involve genomics (the study of the genome), transcriptomics (the study of the set of RNA molecules in one cell or a population of cells, it can be focused on specific classes of RNAs as mRNAs and microRNAs), and epigenomics (the study of the epigenetic marks, mainly methylation/demethylation of cytosines and/or methylation/ demethylation, acetylation/deacetylation of histones). Interesting studies have been recently published and others are currently underway or close to publication. Nemati et al. (2018) applied GBS to solve the problem of the origin of saffron. In GBS, genomic DNA is digested by using restriction enzymes and, subsequently, the generated fragments are amplified via PCR and then sequenced to discover polymorphisms among several samples. As reported earlier, applying this approach the authors obtained interesting evidence supporting an autopolyploid origin of the triploid species. Jain et al. (2016) applied transcriptomic analysis to gain insight into apocarotenoid biosynthesis/accumulation. Transcriptome analysis was performed on RNAs from five different tissues/organs of C. sativus (stigmas, stamens, tepals, leaves, and corms) evidencing the differential expression of transcripts encoding for transcription factors (MYB, MYB related, WRKY, C2C2-YABBY, and bHLH) important for secondary metabolites biosynthesis. A first epigenomic study has been carried out by our research group and is currently under revision. In this study, five accessions characterized by different phenotypes related to flower traits have been analyzed by applying a methylation-sensitive restriction enzyme-sequencing approach. Among the others, the presence of a high number of differentially methylated DNA regions have been detected. Some of these regions host genes encoding for transcription factors important for the flowering process. Clearly, one of the most important goal for any crop of interest for agriculture is the availability of the complete genome sequence. Concerning this point, it is important to note that some initiatives are currently in progress. Sequencing the genome of saffron is not an easy task; in fact, saffron genome is really big (more or less 10.5 Gb), is characterized by a high percentage of repeated elements; and it is a triploid genome. In this respect, the saffron genome can present similar challenges as for other similar polyploidy big genomes (durum wheat or bread wheat). While raw data representing the triplets of chromosomes 1 and 2 have been recently submitted to online available databases, in order to quickly have the whole genome sequence, the realization of organic initiatives, as international consortia, among research groups working on this species, is highly desirable.

Acknowledgements The authors thank the European Commission for funding provided through the AGRI GEN RES 018 action (CrocusBank Project) and the COST Action FA1101 (Saffronomics). Support and cooperation of the Bank of Plant Germplasm of Cuenca (WSCC), headed by Dr Marcelino De-Los-Mozos, are also acknowledged.

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721. Soffritti, G., Busconi, M., Sa´nchez, R.A., Thiercelin, J.M., Polissiou, M., Rolda´n, M., et al., 2016. Genetic and epigenetic approaches for the possible detection of adulteration and auto-adulteration in saffron (Crocus sativus L.) spice. Molecules 21 (3), 343. Springer, N.M., Schmitz, R.J., 2017. Exploiting induced and natural epigenetic variation for crop improvement. Nat. Rev. Genet. 18, 563
575. Torelli, A., Marieschi, M., Bruni, R., 2014. Authentication of saffron (Crocus sativus L.) in different processed, retail products by means of SCAR markers. Food Control 36, 126
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1684. Walker, A.R., Lee, E., Bogs, J., McDavid, D.A.J., Thomas, M.R., Robinson, S.P., 2007. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 49, 772
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Chapter 14

Tissue and cell culture of saffron Nasrin Moshtaghi Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 14.1 Introduction 14.2 Tissue culture of monocotyledons 14.3 Micropropagation of saffron 14.3.1 Explant preparation 14.3.2 Propagation 14.3.3 Acclimation 14.4 Callus and cell culture 14.5 Direct organogenesis

14.1

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14.5.1 Generating adventitious shoots 14.5.2 Microcorm production 14.6 Somatic embryogenesis 14.7 Protoplast culture 14.8 Stigma-like structure 14.9 Conclusion References

234 235 236 239 241 243 243

Introduction

Plant cell and tissue culture is a common term for culturing plant protoplasts, cells, tissues, and organs at in vitro conditions. This method is based on totipotency theory, which means that any part of plant such as tissue, organ, or even cells are able to regenerate new organs or somatic embryo in defined culture medium (Pierik, 1968). This technology started with the Haberland proposition in 1902 regarding cell regeneration potential at the beginning of the 20th century. Haberland proposed that methods should be developed for separating and culturing plant tissues under in vitro conditions. He noted that if the medium and nutrients of cultured cells are manipulated, cells will repeat the growth stages of a whole plant (Pierik, 1968). The discovery of auxin by Went and cytokinins by Skoog was the basis for the first successful in vitro plant culture by Gautheret (1934) and later on by Nobe´court (1939). White (1943) reported the first successful culture of carrot and tobacco callus. Plant tissue culture methods have been introduced as a suitable tool for propagation of many plant species including geophytes such as saffron (Plessner and Ziv, 1999). Different in vitro culture methods including whole-plant culture (such as seed and seedlings), embryo culture (such as immature embryo), organ culture (such as meristem), stem shoot, root, leaf, anther, cell suspension (cell suspension culture), and single-cell and protoplast culture have been developed. Cytological studies have shown that saffron is a triploid plant (2n 5 3X 5 24) and therefore is sterile. Triploid properties make the sexual reproduction problematic but vegetative reproduction is still possible. In fact, saffron is only reproduced by new corms and grows as a slow geophyte plant (Mathew, 1982). The plant is merely reproduced by vegetative methods so that only three to four cormlets are produced in each production season (Fernandez, 2004). The production rate of daughter corms is low in natural conditions. This leads to limitations in accessing materials needed for cultivation (Plessner et al., 1990). Creating new farms is only possible through planting the corms and the corms remain in the soil for a long time (5
7 years). The selected corms should be healthy and free of any disease/damage (Ebrahimzadeh et al., 1998, 2004). Saffron is attacked by fungal and virus pathogens residing in corms that may affect the crop under special conditions. Pathogens remain activated when the corms are brought out of the soil for replanting, which can lead to leaf and corm rotting, necrosis, reducing the flowering or even stopping growth (Plessner et al., 1989; Plessner and Ziv, 1999). Therefore, the corms should be disinfected with fungicides for fungi, but this is not effective for defending against viruses and internal pathogens. However, due to failings in following the cultivation principles such as selecting fertile land, big and healthy corms, weed control, adjusting planting depth, or adding manure or mulch, farmers have to replant their farms every 5
7 years. Thus, providing and selecting favorable corms for cultivation is crucial in order to extend the cultivation area. To ensure the future of saffron, cultivation techniques need to be improved. Any attempt to modernize saffron cultivation needs to be effective in the mass production of corms free of diseases Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00014-9 © 2020 Elsevier Inc. All rights reserved.

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and tissue culture can help in this regard. Today, using tissue culture methods and micropropagation seem to be necessary for mass and free pathogen reproduction of bulbous plants such as saffron (Rajabpoor et al., 2011). In addition, tissue culture can be a very effective and useful method for genetic modification and producing new germplasm of saffron as the plant is sterile and conventional breeding methods cannot be used easily. Protoplast culture, anther culture, and creating changes in germplasm can only be used as saffron breeding methods if in vitro culture methods and its regenerations are optimized. Tissue culture is a useful method for large-scale production of disease-free plants such as saffron using different media with different ratios of auxin and cytokinin (Chen et al., 2003; Chrungoo and Caiola, 1987; Ding et al., 1981; Fakhrai and Evans, 1990; Ilahi et al., 1987; Majourhay et al., 2007; Plessner et al., 1990; Sheibani et al., 2007).

14.2

Tissue culture of monocotyledons

Saffron is a monocotyledon plant from the Iridaceae family. Using bulb and corms of monocotyledons at in vitro culture is not an easy task. Contamination is the biggest problem in their micropropagation especially through their underground organs such as corms, bulbs, rhizomes, and tubers, which are used as explants. The size of underground organs, physical damage, and dormancy are other problems affecting tissue culture studies. Gautheret (1959) first noted that only 10 monocotyledon plants among 100 species are suitable for tissue culture. Partanen (1963) also emphasized the recalcitrant nature of monocotyledons for in vitro culture. The reason is that stems of monocotyledons lack cambium and new vascular differentiation is stopped after growth due to apical meristems, which significantly limits the regeneration possibility. Schenk and Hildebrandt (1972) reported the importance of culture medium and techniques for inducing and developing culture systems in monocotyledons. They found that high levels of auxin and low levels of cytokinin hormones should be applied in monocotyledon plant cell culture. As mentioned before, all cells of a plant body are able to create a new plant or to regenerate and therefore different explants such as corm, terminal and lateral shoots, leaves, nodal tissues, and flowering organs are used for in vitro establishment of saffron (Plessner and Ziv, 1999). Plant cell regeneration is possible through somatic organogenesis and/or embryogenesis in saffron; corms can be regenerated both from somatic organs and embryos (Sheibani et al., 2007; Zeybek et al., 2012). Plant organogenesis is a stage in which cells and tissues produce unipolar structures called shoots and create a vascular system that is connected to the vascular system of the mother tissue. Somatic embryos have bipolar structures containing root and stem, and their vascular system is closed and independent of the vascular system of the mother tissue (Ehsanpoor and Amini, 2000). They both may be produced either directly on the tissue of the mother plant or indirectly through the callus phase (Lagram et al., 2016; Sheibani et al., 2007; Zeybek et al., 2012).

14.3

Micropropagation of saffron

Generally, propagation of saffron is done by corms as the flowers are sterile and are not able to produce viable seeds. Corms survive for a single season and produce up to five cormlets, which eventually give rise to new plants (Deo, 2003). Reproduction of saffron is dependent on human work because the corms need to be manually dug up, broken apart, and replanted. Therefore, the natural propagation rate of most geophytes including saffron is relatively low. Also all of the saffron accessions are genetically uniform so the germplasm variation is narrow, which creates a challenging task when it comes to breeding. Therefore, selection of larger corms for producing more flowers is a way to increase the yield (Agayev et al., 2009). Moreover, in vitro culture of saffron is important for the propagation of this economic plant and to produce larger corms. Conventional propagation methods are very slow because expansion of the cultivation area of saffron depends on producing daughter corms by mother corms. Propagation of daughter corms via tissue culture such as clonal propagation, organogenesis, and somatic embryogenesis in callus cultures followed by generation of shoots/plantlets represents a potential effective propagation method (Blazquez et al., 2004a; Sharma et al., 2008). There are different reviews on tissue culture of saffron (Ascough et al., 2009; Plessner and Ziv, 1999; Sharma and Piqueras, 2010). Different experiments were designed to propagate the saffron in vitro culture using direct or indirect shoot induction or plantlet regeneration through somatic embryogenesis followed by microcorm production. Explant preparation, propagation, and acclimation are the main steps for proliferation of saffron.

14.3.1 Explant preparation Different explants such as corm segments, leaves, and floral parts of saffron have been used for micropropagation, but corm segments, which have eye buds, usually respond better than other explants for producing shoots (Karaoglu et al., 2007). Different explants are used for callus production, somatic embryogenesis, protoplast culture, and stigma-like structures (SLS) (Plessner and Ziv, 1999; Sharma and Piqueras, 2010).

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Sterilizing the explants is difficult because usually the plant samples are brought from the field to the lab where plant parts, especially corms, are contaminated with different microorganisms. Surface contaminations could be removed easily using standard sterilizing techniques and disinfectants such as mercuric chloride, ethanol, and sodium hypochlorite. However, it is very difficult to remove endogenic contaminations even under the best sterilization conditions. The spores or dominant microorganisms occurring as endogenic contaminants are usually resistant to disinfectants. Hence, different treatments by antibiotics and fungicides are needed for sterilization and inhibition of the growth of endogenic contaminants even 3
5 weeks after culture. It is normally not possible to achieve complete sterilization because of high percentages of endogenic contaminations. Parray et al. (2012) used the corms as explant. At first, the corms were thoroughly washed with detergent Extran (0.5%) and Tween-20 (surfactant) followed by rinsing with double distilled water. Subsequently the corms were surface-disinfected with 70% ethanol for 1 minute followed by 0.5% HgCl2 (w/v) for 6 minutes and washed five times with distilled water. Karaoglu et al. (2007) used different explants including floral and corm segments. In their report, washing in running water, sulfuric acid, and fungicide treatments was used to sterilize corms.

14.3.2 Propagation The propagation rate in saffron is very important for its micropropagation. Regeneration of plants from isolated protoplasts is limited (Darvishi et al., 2007; Isa et al., 1990). Callus induction followed by somatic embryogenesis or organogenesis is another way of regeneration of saffron. Embryogenesis is usually preferred due to its high regeneration (Blazquez et al., 2009; Ding et al., 1981, 1979). Direct shoot regeneration from different explants of saffron is more efficient than somatic embryogenesis. Direct shoots have been generated from different explants such as apical buds, lateral buds, small corms (Fig. 14.1), and ovaries (Plessner and Ziv, 1999; Sharma et al., 2008).

FIGURE 14.1 Explants from corms (apical bud, lateral bud, and small pieces of corm). From Hagizade, A., 2016. Optimizing Propagation of Saffron (Crocus sativus L.) Corms by In Vitro Direct Regeneration (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

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Direct shoots are genetically uniform because they do not pass through the intermediate callus phase. Different media such as MS, SH, B5, and 1/2 MS media with different combinations of growth regulators were used for multiple shoot induction and producing cormlet at in vitro culture (Homes et al., 1987; Majourhay et al., 2007; Sharifi et al., 2010; Sharma et al., 2008). During shoot/plantlet development experiments, it has been found that in vitro developed shoots have a tendency to swell at the base and produce the cormlets or microcorms (Sharma et al., 2008). A corm can be divided into at least three explants, so the more cormlets produced from each explant the higher the rate of propagation. A combination of one auxin and one cytokinin is usually used for propagation though the ratio of cytokinin to auxin is .1. For example, in the report of Karaoglu et al. (2007) different explants including floral and corm segments were used on different media with different growth regulators to produce shoots. New corms were produced on MS medium supplemented with 2 mg L21 6-benzylaminopurine (BAP) and 0.5 mg L21 NAA after 6 months of culture.

14.3.3 Acclimation Acclimation of the cormlets produced in in vitro culture is very important. Since only corms with a weight over 5 g can produce flowers in the first year, increasing the corm weight and diameter are the main factors at in vitro culture and subsequently in acclimation. There are three concerns to be considered in saffron micropropagation: (1) sustained multiplication of shoots from tissue culture-derived shoots, (2) development of large microcorms, and (3) field evaluation of microcorms for agronomic performance (Sharma and Piqueras, 2010). In one report on the evaluation of microcorms under field conditions (Parray et al., 2012) the cormlets with 2.5 g weight were planted in clay loam soil in a greenhouse at 20 C 6 2 C. A flowering rate of 25% was observed using the cormlets, while for 2 g cormlets the rate of flowering was only 19% (Parray et al., 2012).

14.4

Callus and cell culture

Callus is a collection of unorganized cells produced by dedifferentiation on explants. Different explants such as meristemic and nonmeristematic parts of plants can produce callus. Callus induction is a process affected by explants, media, and growth regulators especially auxins. The goals of callus culture are metabolite production, embryogenesis, organogenesis, and SLS production. Some researchers have shown that callus and cell cultures can produce low concentrations of coloring metabolites such as crocin and crocetin in compared to the natural stigmas or SLS cultures (Hori et al., 1988; Zeng et al., 2003). Zeng et al. (2003) reported that crocin content in saffron callus was only 0.24% compared to 14.30% in natural stigmas and 6% in SLS cultures. Browning of cultures usually is a limited factor for producing metabolites through cell culture (Visvanath et al., 1990). Generally, two-stage culture of cells is better than one-stage culture because the optimum conditions for cell growth are different from those for metabolite production. A combination of NAA and BAP in B5 medium plus casein hydrolysate was shown to be the best for cell growth, while a combination of IAA and BAP was suitable for crocin production (Chen et al., 2003). Darkness and temperature of 22 C were the optimum physical conditions for crocin production (Chen et al., 2003). Some rare elements such as La1 and Ce1 may enhance crocin production in cell culture. These elements in low concentrations act like heavy metals to elicit secondary metabolite production (Chen et al., 2004). Further experimentation is needed to optimize factors affecting cell growth, metabolite production at in vitro cultures, and bioreactors. Most of the research is focused on crocin production due to its anticancer properties (Escribano et al., 1996). Also, saffron corms are rich in specific proteoglycans, which inhibit the growth of human tumor cells. These proteoglycans are also synthesized in callus cultures developed from saffron corms (Escribano et al., 1996). Raja et al. (2007) reported the highest rate of callus formation in MS medium containing 1 mg L21 BAP and 1 mg L21 2,4-D. In addition, they used MS medium containing 2.25 mg L21 BAP and 0.1 mg L21 2,4-D for somatic embryo formation. The highest rate of matured and germinated embryo was observed in 1/2 MS medium containing 1.75 mg L21 ABA, 0.5 mg L21 BAP, and 20 mg L21 GA3. The highest number of small spherical microcrom was obtained in MS medium containing 4 mg L21 BAP and 0.5 mg L21 NAA with 9% sucrose after 8
10 weeks. Vatankhah et al. (2012) found that the lifting time of corms, type of culture medium, and hormone treatment affected the formation of nodular callus. For this purpose, corms were collected both in early June and October and placed on MS, LS, and B5 culture media containing 1 mg L21 2,4-D, and 0.5, 1, and 4 mg L21 kinetin, respectively. The results showed that the highest percentage of nodal callus production was produced from corms harvested in June and cultured on B5 medium with hormone treatment of 1 mg L 2,4-D along with 4 mg L21 kinetin. The obtained nodal calli were transferred to MS liquid culture medium without hormones, which formed shoot-like masses with different

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shapes after subculture (was not subcultured for 2.5 months). Fifty nine percent of the shoot-like masses formed nodal callus in 1/2 MS medium containing 1 mg L21 of BAP and NAA from which only one node turned to shoot. Ziaratnia et al. (2012) applied different hormone treatments to saffron to study factors affecting callugenesis. To do this, 72 combinations of growth regulators including 2,4-D and NAA (2, 4, and 8 mg L21) alongside the BAP and kinetin (1, 4, and 8 mg L21) were applied in MS and B5 culture media to induce callus formation. Results showed that the combination of 2,4-D (2 mg L21) with BAP (8 mg L21) in B5 medium is the best callugenesis treatment. Mirjalili and Poorazizi (2012) noted that the aim of studying embryogenesis and culturing apical bud in saffron is determining the best range of growth hormones, sucrose, and temperature for embryogenesis and branching of saffron. Also, treatment containing 45 mg L21 sucrose showed the highest branching rate and 4 C was the best temperature for embryogenesis. Vahedi et al. (2012) established somatic embryogenesis as an effective method for fast and mass reproduction of saffron. They studied the effect of two growth regulators of 2,4-D in concentrations of 1, 2, and 4 mg L21 and kinetin in concentrations of 0.5, 1, 4, and 8 mg L21 in MS medium on callus induction of apical and lateral meristems of saffron corms. The results showed that the first signs of callus induction on explants of lateral meristems appeared after 60 days and developed after 45 days for apical meristems in MS medium containing 2 mg L21 2,4-D and 0.5 mg L21 kinetin and explants obtained from apical meristems produced more callus (44%) during less time (45 days) compared with lateral ones (60 days after placing the explant and about 16%
32% callugenesis). Sharafzadeh (2012) placed leaf explants of saffron in MS medium containing 0.1, 0.2, 0.3, 4.0, and 0.5 mg L21 BAP and 1, 1.5, 2, 2.5, and 3 mg L21 2,4-D. Green calluses formed in 2.5 mg L21 2,4-D and 0.3 mg L21 BAP. Yasini et al. (2013) used 36 growth regulator compounds including 2, 4, and 8 mg L21 of 2,4-D and NAA along with 1, 4, 8 mg L21 BAP and kinetin in MS medium to induce callus. Also, 24 hormonal combinations including 0.5 and 1 mg L21 of 2,4-D and NAA along with 0.2, 0.5, and 1 mg L21 BAP and kinetin in MS medium were used to induce brittle callus formation. The results showed that 8 mg L21 of NAA alongside 1 mg L21 of BAP was the best treatment for callugenesis in MS medium. The best treatment for producing brittle callus was 1 mg L21 of 2,4-D and 0.2 mg L21 of kinetin. Sajjadifar and Pazhoohande (2015) studied the effect of 36 different hormone compounds in dark conditions and nine different ones in cold treatment conditions (4 C) using different explants (corm, leaf, leaf end, and leaf scale) on callugenesis. Only corm explants responded favorably to callugenesis in MS medium with 1 mg L21 IBA and 2 mg L21 BAP in both dark and cold conditions. The highest percentages of regeneration with microcorm formation (20%) was obtained in MS medium with hormone combination of 0.3 mg L21 TDZ, 1 mg L21 BAP, 2 mg L21 IBA, and 0.01 mg L21 GA3 in cold conditions. Moghbeli et al. (2015) studied the effect of different growth regulators of BAP and 2,4-D on callus induction and embryogenesis in saffron. Corm explants were cultivated on MS medium containing BAP with concentration of 2 mg L21 and 2,4-D with concentrations of 0.5, 1, 3, and 5 mg L21 for callugenesis. Callus generated on MS medium containing BAP with constant concentration of 3 mg L21 and NAA with concentrations of 0, 0.1, 1, 3, and 5 mg L21 were subcultured with the aim of regeneration and embryogenesis. Callugenesis results showed that increasing the concentration of 2,4-D from 0.5 to 5 led to larger calluses. In addition, increasing the concentration of NAA reduced the embryogenesis of calluses. In fact, the best medium for embryogenesis was the medium containing 3 mg L21 BAP without NAA. Lagram et al. (2016) obtained saffron callus from corm explant in MS medium containing 1 mg L21 of both 2,4-D and BAP hormones. In addition, results showed that regeneration of aerial organs from callus happened in MS medium containing 1.5 mg L21 BAP and MS medium containing 8 mg L21 BAP alongside 2 mg L21 NAA after 2
4 months and there was no significant difference between culture media. Then, shoots were cultivated on 1/2 MS medium containing 1 mg L21 of both 2,4-D and BAP hormones as well as 3% and 5% sucrose. The average number of 0.39 and 0.79 microcorms formed in both concentrations of sucrose after 2 months, respectively, and there was no significant difference between sucrose concentrations.

14.5

Direct organogenesis

Different factors such as plant species, genotype, explant size and type, age, and the salts of culture medium affect organogenesis. There have been considerable advances in tissue culture of plants after discovering plant hormones and the morphogenesis hypothesis using auxin to cytokinin ratio is the basis of morphogenesis control (Hasandokht and Ebrahimi, 2006).

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14.5.1 Generating adventitious shoots Adventitious shoot is a kind of shoot not formed in its main location (at the end of stem and node); for example, it is formed in the internodal part of explants or other parts. The reason for adventitious shoot formation is dedifferentiation and redifferentiation of some cells. Adventitious shoots can be generated either directly from explants containing external parts of phloem and surface combium or indirectly from the surface of callus. Direct shoot formation has low or no variety while the indirect shoot formation is more diverse. Sometimes, callus generates adventitious shoots after several subcultures. Adventitious root is also a kind of root formed in a location other than its main location. Auxin is necessary for the formation of adventitious roots while cytokines usually prevent rooting in the presence of auxin as they accelerate cell division and prevent differentiation. Some advantages of direct adventitious shoots are: 1. Reproduction rate of the method is high. 2. Most characteristics of the plant are usually maintained. However, there is the possibility of changing and variation in some cells. 3. Time period to flowering is reduced (Hasandokht and Ebrahimi, 2006). Direct shoots without intermediate callus induction phase is preferred over indirect shoots or somatic embryogenesis for saffron propagation. Direct shoots have been generated from small corms, apical bud, lateral buds, and ovaries (Plessner et al., 1990; Sharma et al., 2008; Fig. 14.1). Less time is usually required for generating direct shoots compared to indirect ones. Type of explant, growth regulators, and other factors such as temperature affect the organogenesis. The first report of direct shoot generation was from corms (Homes et al., 1987). Shoot induction from apical buds was induced by cytokinins such as kinetin and zeatin at 15 C or 20 C (Plessner et al., 1990). BA is the most effective cytokinin in direct shoot induction (Majourhay et al., 2007; Sharma et al., 2008). Sharifi et al. (2010) reported that TDZ at low concentration below 10 μM was more effective than BA. Moreover, high concentration of sucrose (50 g L21) was better than low concentration (30 g L21) for shoot induction (Hagizade, 2016; Sharma et al., 2008). Saffron ovaries have been used to generate callus, shoots, and SLS. The ratio of cytokinin to auxin seems to be the most critical factor leading to organogenesis or dedifferentiation. The absence or low concentrations of IBA or NAA, alongside BA lead to direct shoot generation from ovaries (Darvishi et al., 2007; Sharma et al., 2008). Callus induction occurs when the ratio of auxin to cytkinin is high. Also, the equal ratio of BA and NAA causes the SLS from ovaries (Castellar and Iborra, 1997). Efficient micropropagation of saffron depends on the number of multiple shoots. In the research by Sharma et al. (2008), the maximum number of shoots of four per culture was induced at 14 mg L21 BA 1 3 mg L21 IBA 1 50 g L21 sucrose, but this frequency is still low. In another study, the maximum number of shoots induced on corm segments was 2.62 in SH medium supplemented by 3 mg L21 BA and 1 mg L21 of NAA (Hagizade, 2016; Fig. 14.2). FIGURE 14.2 Direct shoot generation from saffron corm segments. From Hagizade, A., 2016. Optimizing Propagation of Saffron (Crocus sativus L.) Corms by In Vitro Direct Regeneration (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

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Piqueras et al. (1999) used 2 mg L21 of BAP and 0.1 mg L21 of NAA for growth and propagation shoots from corm explants. Zeybek et al. (2012) studied the effect of different levels of 2,4-D (0.1 and 1 mg L21) in combination with BAP (1, 1.5, and 2 mg L21) on direct shoot formation. Combination of 0.1 mg L21 of 2,4-D with 2 mg L21 BAP caused high percent of shoot formation but using 1 mg L21 of BAP alone resulted in more shoots being produced per explant. Also, cormogenesis from shoots at different levels of IBA (0, 1, and 2 mg L21) was studied and the results showed that 1 mg L21 of IBA and 5% sucrose leads to a high percentage of cormogenesis (52%) with the average number of 1.04 microcorm per explant. Karaoglu et al. (2007) examined plant regeneration from meristematic regions of corms by direct organogenesis. In their experiment, apical and lateral buds of corms were cultured as explant on MS and 1/2 MS media. MS medium containing 6 mg L21 BAP led to the best result in terms of shoot and leaf formation rates. In addition, the lowest shoot formation rate occurred in MS medium containing 1 mg L21 BAP and MS medium containing 10 mg L21 BAP. Data shows that MS medium containing 1 mg L21 IAA has the best microcorm formation rate (76.7%) with an average microcorm number of 1.74 per explant (Karaoglu et al., 2007). Morata et al. (2013) used corms maintained at low temperature as explant source for in vitro saffron reproduction. Corms maintained in cold conditions geminated well in a culture medium containing high cytokinin (i.e., 5 mg L21 of BAP or 1 mg L21 TDZ and 0.05 mg L21 NAA). Simona et al. (2013) performed two experiments, direct organogenesis stimulation (forming adventitious shoots using lateral and apical buds) and indirect organogenesis stimulation (callus formation using corm parts except shoots). The results showed that the mixture of 1 mg L21 2,4-D and 1 mg L21 BAP is suitable for direct regeneration while the mixture of 0.25 mg L21 2,4-D and 1 mg L21 BAP is more suitable for indirect regeneration. The range of regeneration using indirect in vitro organogenesis is limited. The callus induction rate was 5% and the whole parts of callus were not embryogenic. In addition, many embryos failed to evolve. The best result was achieved in an average of three microcorms per explant through direct organogenesis. Mir et al. (2014) cultivated explants of terminal shoots in different media with different concentrations of growth regulators. In their experiment, different concentrations of BAP (2.22, 22.2, 4.44, and 44.4 μM) and NAA (10.8, 16.2, 21.6, and 27 μM) were used for regeneration and also different concentrations of BAP (0.5, 1, 1.5, and 2 mg L21) were used for corm reproduction. Microcorm formation was observed in all combinations of medium. Most microcroms were created in MS medium containing 2 mg L21 BAP and 0.5 mg L21 NAA with an average number of 10.2 microcorms per explant. Also, culture medium was not affected by the presence or absence of light for forming microcorm and their growth. The highest number and longest shoots were obtained in MS medium containing 21.6 μM NAA and 22.2 μM BAP. Sivansan et al. (2014) studied shoot and microcorm regeneration from corm explant of Crocus vernus. Corms were cultured in SH medium supplemented by 0.5, 1, 2, and 4 mg L21 of 2ip, BAP, and kinetin alone and/or in combination with 0.5 mg L21 NAA. BAP was the best cytokinin for shoot formation among three cytokinins. When BAP was used with 0.5 mg L21 NAA, the number of shoots per explant was higher than BAP along with 1 mg L21 NAA. The highest percent of shoot formation per explant was obtained from SH culture medium containing 2 mg L21 BAP and 0.5 mg L21 NAA. Abundance of induced microcorms was affected by sucrose concentration. The highest number of microcorms was obtained from the medium containing 6% sucrose and the average number was 6.1 microcorms per explant. Shoot generation using SH and MS media as well as NAA and BAP compounds can activate and produce more shoots than natural conditions and convert them into corms. The best culture medium for shoot generation was SH culture medium supplemented by 1 mg L21 NAA and 3 mg L21 BAP. If the shoots were subcultured in their previous medium for 3 months, their base would be swollen and cormlets would be produced. The average number of cormlets per explant was 2.11 corms for the best subculture (Hagizade, 2016).

14.5.2 Microcorm production Generation of adventitious shoots and somatic embryogenesis is highly considered for saffron propagation and cormogenesis. In natural propagation of saffron, the cormlets develop on the mother corms and these cormlets grow for 3
4 years to give rise to the next generation of cormlets. At in vitro culture, produced shoots have a tendency to swell at the base and produce microcorms (Gui et al., 1988; Hagizade, 2016; Sharma et al., 2008). The microcorms are generated in a short time and are easy to transport and store at low temperature for germplasm storage. The corms with adventitious shoots, which are rooted in medium without growth regulators, also give rise to microcorms. Some treatments such as

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ethylene, microsurgery of apical buds, modification of salt concentration in media, growth regulators, growth retardants (such as paclobutrazol and imazalil) and delaying in subcultures caused inducing of microcorm production (Hagizade, 2016; Piqueras et al., 1999; Plessner et al., 1990; Sharma et al., 2008). Sharma et al. (2008) transferred the in vitro shoots to 1/2 MS culture medium, MS medium supplemented by 3 mg L21 BAP, and 80 g L21 sucrose, and 1.89 microcorms per explants was observed. They showed that sucrose is very important for microcorm induction. Higher sucrose concentration (6%
9%) is preferred for microcorm production (Hagizade, 2016; Raja et al., 2007; Sharma et al., 2008) though it is not effective in all plants. Sucrose can provide the required energy for microcorm induction, and thus using mannitol as a replacement of sucrose did not induced the microcorm production (Sharma et al., 2008). Lower salt concentrations (e.g., 1/2 MS salts or SH salts) improve microcorm induction (Hagizade, 2016; Raja et al., 2007). Growth regulators IBA, BA, NAA, and ABA affect microcorm induction. Sharma et al. (2008) used 1/2 MS medium supplemented by 3 mg L21 of BA and produced 1.89 microcorms (1.18 g average weight) per shoot. BA alone or in combination with other growth regulators is the most important growth regulator for cormogenesis (Piqueras et al., 1999; Sharma et al., 2008). ABA, which is related to senescence, inhibits cormogenesis (Chichiricco and Grilli Caiola, 1987). A temperature range of 10 C
25 C was tested for cormogenesis in saffron resulting in suitable corm induction at 20 C (Bhagyalakshimi, 1999; Milyaeva et al., 1995; Plessner et al., 1990; Sharma et al., 2008). The effect of light in corm induction of saffron is not clear and cormogenesis occurred in partial and continuous light. More research is needed on the role of light and its interaction with temperature and growth regulators (Sharma and Piqueras, 2010). In the study of Hagizade (2016) the shoots were transported to new 1/2 MS medium without hormones containing 60 g L21 sucrose for vascular system differentiation and accelerating starch transportation to basal part of shoots, when cormogenesis occurred. The average number of cormlets per explant was 2.62, which is more suitable than 3
4 daughter corms that produced in the field. At in vitro conditions, each corm is cut into three slices and each slice could produce 2.62 cormlets which were disease-free. In their study, unlike most studies that use sucrose for increasing osmotic pressure and microcorm production, the study induced drought stress that converted shoots into cormlets. Therefore, this treatment can reduce the cost of cormogenesis as no (or little) sucrose is required in the process. The weight of in vitro microcorms is very important because the larger corms give rise to larger and more cormlets in vivo that bear more flowers (Agayev et al., 2009; Gresta et al., 2008). In their protocol for cormogenesisSharma et al. (2008) reported that the weight of cormlets were 1.18 g after 9 months of culturing, buds but the daughter corms, which were produced in field conditions, had 1.2 g average weight after 22 months (Chahota et al., 2003). Sucrose can increase the weight and size of corms. In the study of Sargazi-Moghadam (2019), using 1/2 MS medium with 6% sucrose was better than 3% sucrose to increase the diameter of corms as cormlets with weight of 7.4 g and diameter of 1.22 cm was produced (Fig. 14.3). In the study of Sargazi-Moghadam (2019), using of 6% sucrose in 1/2 MS medium was better than 3% sucrose for increasing the weight and diameter of cormlets (cormlets with weight of 7.4 g and diameter of 1.22 cm, Fig. 14.3). Parray et al. (2012) developed a complete protocol for saffron cormlet production in in vitro conditions. They used 1 /2 MS medium supplemented by TDZ, IAA, and high sucrose for cormlet production. They produced 70 cormlets from one corm slice, with average cormlet weight of 2.5 g. The cormlets were planted in clay loam soil in a greenhouse at 20 C 6 2 C. The rate of flowering of the cormlets was 25%. However, the flowering rate declined to 19% when the weight of the cormlets was 2 g.

14.6

Somatic embryogenesis

Flowering plants can create somatic embryos that are developed from somatic cells and tissues in addition to fertilized ovule development. Somatic embryogenesis occurs when an embryo is generated from haploid or diploid nonreproductive cells. In fact, somatic embryogenesis is a process in which somatic cells are differentiated and form bipolar structures including root and stem base. Somatic embryos are similar to reproductive embryos and are able to develop to whole plants. Somatic embryo is generated by endosperm tissue, meristematic cells, and/or leaf edge cells in many plant species. Somatic embryo formation is one of the most important ways for regeneration through in vitro tissue culture, which is performed, either directly from a cell and/or a differentiated cell-like stem, or indirectly from callus culture (Ehsanpoor and Amini, 2000). Somatic embryogenesis was first produced in cellular suspension cultures of carrot in 1958 (Pierik, 1968). The first report on successful induction of callus and regeneration of intact plantlets in saffron was from corm explants (Ding et al., 1979, 1981; Sharma and Piqueras, 2010). They used IAA and 2,4-D for their culture media.

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FIGURE 14.3 In vitro cormlet development of Crocus sativus L. (A) multiple shoots on SH 1 BAP (3 mg L21) 1 NAA (1 mg L21) 1 3% sucrose, (B) growth of shoots on SH 1 BAP (3 mg L21) 1 NAA (1 mg L21) 1 3% sucrose, (C) producing cormlets on 1/2SH 1 IBA (1 mg L21) 1 3% sucrose, (D) growth of cormlets on 1/2MS 1 IBA (1 mg L21) 1 6% sucrose, and (E and F) vegetative growth under greenhouse conditions. From SargaziMoghadam, Z., 2019. The Effect of Induced Mutation on Saffron (Crocus sativus L.) Corm Propagation at In Vitro Culture (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (In Persian).

Somatic embryogenesis phases are similar to generative embryogenesis, and include spherical, heart-shaped, knifeshaped, and cotyledonary phases in dicotyledons, and spherical, scutellum (horn-shaped), and cotyledonary phases in monocotyledons (Denis et al., 1991). Embryogenesis includes the following steps: 1. Induction: Induction is performed by putting explants in media with high concentrations of auxin, which prepares cells to enter the dedifferentiation phase. 2. Differentiation: The differentiation phase occurs after induction in which explants are transferred to the medium in the presence of cytokinin and low concentration of auxin. In this phase, prepared cells tend to go back to the differentiated phase so that they can proliferate with plant hormones in culture medium and generate somatic embryos. 3. Maturation: In this step, somatic embryos are transferred to a culture medium with low concentration of hormones. Somatic embryos grow through phases similar to embryogenesis in gymnosperms, monocotyledons, and dicotyledons (Thrope, 1995). Somatic embryos are sometimes different from generative ones. Generative embryos result from fusion of two gametes and sexual reproduction, while somatic embryos result from somatic reproduction and are usually produced by somatic cells. The generative embryo supplies nutrient suspensor and embryo growth regulators and it also has endosperm, cotyledon, and seed coat. Somatic embryos use nutrients and hormones provided by culture medium; and they have no endosperm, suspensor, and shell and cotyledons are smaller. However, it is sometimes observed that the suspensor connects the embryo to the explant and feeds on it (Hasandokht and Ebrahimi, 2006). Another important difference is that generative embryos are dormant after the formation of embryonic bases or terminal meristems while somatic embryos do not enter this step and embryogenesis from basic cells to the formation of seedling is started immediately after being placed on culture medium and continues without any break (Kumar, 2003). Embryogenic cells creating a visible embryo have nearly similar characteristics in all in vitro culture systems; these characteristics include small size, compressed cytoplasmic content, big nucleus, small vacuole, and low starch grains.

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Molecular and histological studies emphasize high RNA synthesis and intense metabolism activity in these cells (Ehsanpoor and Amini, 2000). In direct embryogenesis, the embryo is directly generated from a cell or a tissue without callus formation. Basically, direct somatic embryogenesis is occurred in differentiated cells, which has been determined for embryo production earlier. These cells, which are called preembryonic or embryonic, determined cells have been already assigned to embryo development before being separated as explant and they just need growth regulator or suitable conditions to begin cellular division step and expressing embryogenesis. Hypocotyl epidermis, epidermis tissue, and symmetrical nuclei are very suitable materials for this kind of embryogenesis. In the indirect embryogenesis method, somatic embryo induction occurs after the callus induction phase. Somatic embryos are generated through the callus intermediate phase and when cell capacity and capability change. Different kinds of explants such as corms, corm pieces, apical and lateral buds, young ovaries, young leaves, and shoot apices induce callus in saffron (Blazquez et al., 2004a; Sharifi et al., 2010; Sharma et al., 2005). But the callus can be embryogenic and nonembryogenic. Usually 2,4-D is required for induction of embryogenic callus whereas lack of this hormone leads to the development of nonembryogenic calli (Darvishi et al., 2007; Karamian, 2004; Raja et al., 2007; Sharma et al., 2005). 2,4-D alone is not very effective at embryo induction and hence a combination of 2,4-D with kinetin or BA is essential for high frequency of induction (Karamian, 2004). Jasmonic acid (Blazquez et al., 2004b) and TDZ (Sheibani et al., 2007) also improve efficiency of somatic embryogenesis. Whole callus usually do not produce embryos and only some of the regions can do it (Blazquez et al., 2009). Somatic embryogenesis was carried out on different species of Crocus successfully by Verma et al. (2016). Callus induction, somatic embryogenesis, and plant regeneration were initiated in five selected species of Turkish Crocus. The embryogenic calli developed into cotyledonary embryos in media containing (2 mg L21 IAA 1 2 mg L21 TDZ) and 100 mg L21 ABA. In their study Crocus oliveri ssp. Oliveri produced the most embryos per explant (Verma et al., 2016; Fig. 14.4). A high level of sucrose (6%) in hormone-free MS medium (Sheibani et al., 2007) and 1 mg L21 of ABA led to maturation of embryos (Karamian, 2004). Mature embryos were germinated on 25 mg L21 of GA3 (Karamian, 2004). Moreover, the basal parts of the embryos usually swell leading to the formation of microcorms after 3 months

FIGURE 14.4 Plant regeneration through indirect somatic embryogenesis from the leaf explants of Crocus species Crocus oliveri ssp. Welldeveloped globular somatic embryos of C. oliveri (A) developing stages of cotyledonary stages of somatic embryos, (B
D) on the callus from leaf explants after 3 months of culture on MS medium supplemented with 2 mg L21 indole-3-acetic acid (IAA) plus 2 mg L21 thidiazuron (TDZ) and 100 mg L21 abscisic acid (ABA), (E) well-developed cotyledonary stage somatic embryos, (F) A plantlet regenerated from a somatic embryo, and (G) Regenerated plants in the potted soil. From Verma, S.K., Das, A.K., Cingoz, G.S., Uslu, E., Gurel, E., 2016. Influence of nutrient media on callus induction, somatic embryogenesis and plant regeneration in selected Turkish crocus species. Biotechnol. Rep. 10, 66
74.

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(Sheibani et al., 2007). Producing callus in a temporary immersion system increases the fresh weight of embryogenic calli four times compared to those grown on solid media (Blazquez et al., 2004b). The embrogenic callus has a nodular structure and takes around 6 weeks to get a nodular shape. At this stage, proembryogenic structures or proglubular embryos exist on callus that will be developed to globular embryos after 3 weeks in culture, to monopolar after 7 weeks, and to bipolar embryos after 10 weeks in culture (Blazquez et al., 2009). In saffron, oxidative stress followed by reactive oxygen species (ROS) appear to function as components of signal transduction chain required to reprogram gene expression and induce totipotency to gain embryogenic competence by the somatic cells (Blazquez et al., 2009, 2004a). The shoots or plantlets from callus cultures develop in the presence of cytokinins (BA, kinetin), either alone or in combination with auxins such as IBA or NAA (Ahuja et al., 1994; Sharma et al., 2005). The type of nitrogen source is also important for shoot regeneration from nonembryogenic calli. Nitrogen in the form of nitrate (NO32) was more effective than ammonia form (NH41) for induction of shoots (Igarashi and Yuasa, 1994). By studying somatic embryogenesis and seedling regeneration in saffron, Ahuja et al. (1993) found that the best culture medium for somatic embryogenesis is aerial organ meristems and LS medium containing 2 3 1025 M of NAA and 2 3 1025 M of BAP. In addition, mature embryos germinate in 1/2 MS medium containing 20 mg L21 GA3. In addition, microcorm formation happens when a germinated embryo is transferred to 1/2 MS medium containing 2% activated charcoal and 5 3 1025 M of NAA and BAP. Ebrahimzadeh et al. (2000) found that the best culture medium for somatic embryo evolution (from globular phase to embryo) is obtained from aerial organ meristem and LS culture medium containing 2 3 1025 M NAA and 2 3 1025 M BAP. Also, mature embryo germinates in 1/2 MS medium containing 25 mg L21 GA3. By studying somatic embryogenesis induction of saffron using TDZ, Sheibani et al. (2007) concluded that the most effective treatment is TDZ with the concentration of 0.5 mg L21 in which the first embryogenic calluses appeared 8 weeks after explant culturing. Then, embryos were transferred to MS medium without hormone containing 6% sucrose to mature where the matured embryos turned to microcorm after 3 months in 1/2 MS medium. It was reported that the highest number of spherical embryos were formed in 1/2 MS medium containing 0.1 mg L21 NAA. The highest number of mature embryos was observed in 1/2 MS and 0.5 mg L21 BAP, but the continuity of using this hormone was not effective in microcorm formation. One to two mg L21 IBA was the best treatment for the embryo to mature and for microcorm formation (Rajabpoor et al., 2011). Also, somatic embryogenesis was done from upper and lower corm explants using 20 mg L21 2,4-D and 1.0 mg L21 BAP. The results showed that lower corm explants were much more responsive for the percentage of embryo response (Rajabpoor et al., 2007). Sivansan et al. (2012) studied different concentrations of 2,4-D hormones in combination with 0.5 mg L21 TDZ to induce somatic embryogenesis in C. vernus in SH medium. The highest rate of embryogenesis happened in culture medium containing 1 mg L21 2,4-D. Embryos were transferred to SH medium containing 1 mg L21 GA3 to mature and were prepared to develop to corm after maturing and germination. Germinated embryos converted to seedling in a culture medium containing 1 mg L21 GA3 and 6% sucrose. In the direct method, the combination of 2,4-D and TDZ led to somatic embryogenesis in a month, after explant culturing without passing through the callus phase (Hagizade, 2016). Decreased somatic embryogenesis was caused by increasing the concentration of 2,4-D above the optimum level. Somatic embryos prepare for converting into cormlets after shooting in SH medium containing 2 mg L21 BAP and 1 mg L21 GA3 and 6% sucrose. Buds developed after 3 months in the culture medium and then were not subcultured for 3 months. Finally, this stress caused the base of some shoots to swell in the third month and produced cormlets. The average number of cormlets per explant was 2.66 (Hagizade, 2016; Fig. 14.5). The cost of shoot production in vitro cultures is high due to the labor, agar, and sucrose costs and thus alternative methods are required. Using robots as labor works and photoautotrphic micropropagation under high light and CO2 intensity especially after induction of shoots and using liquid media for eliminating agar are alternatives for reducing the cost of micropropagation (Sharma and Piqueras, 2010). Sharma et al. (2005) used cotton bed as a substitute of agar and the cost was reduced by 33.5%. But the frequency of shoot regeneration was low. Maybe agar is suitable for nutrient availability and uptake by saffron cells (Karim et al., 2003).

14.7

Protoplast culture

Advances in plant in vitro culture such as culturing plant cells, tissues, organs, and cells without cell wall (protoplast) have opened new ways to perform the basic genetic research at a cellular level. It also provided a powerful tool for plant basic research to produce, select, and develop advanced varieties of plants. Plant protoplast cultivation and regeneration is one of the effective methods of cell culture that can be used to produce new plant species through fusing

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FIGURE 14.5 Germination of somatic embryos in SH culture medium containing 2 mg L21 BAP and 1 mg L21 GA3 and 6% sucrose. From Hagizade, A., 2016. Optimizing Propagation of Saffron (Crocus sativus L.) Corms by In Vitro Direct Regeneration (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

somatic cells and/or genetic improvement of a plant species by transferring genes (Vasil and Vasil, 1979). Plant protoplast fusion is a biotechnological method resulting in a new somatic hybrid through nuclear or cytoplasmic genome combination to overcome incompatibility between the species. Protoplast fusion is a mix of two different protoplasts. Lack of cellular walls lead to close contact between two protoplasts or more and sometimes leads to protoplast content exchange. Somatic hybridization (nonreproductive hybridization) is performed through protoplast fusion. Somatic hybridization means fusion of two protoplasts so that it can provide different genetic compounds (Ahooran et al., 2009). Gene transferring and modification using this method can be performed in cases such as sexual incompatibility, multiembryo, and male and female sterility. Protoplast fusion is used for intraspecific, interspecific, intrageneric, and intergeneric crosses (Crosser and Ollitrault Olivares-Fuster, 2000). Saffron is a plant reproduced by resting corms and thus it is difficult to improve its agricultural characteristics by conventional methods. Protoplast culture and its hybridization is a suitable tool for improving its agricultural characteristics (Ahooran et al., 2009). The first successful hybridization effort was on tobacco somatic hybridization by Carlson et al. (1972) in the United States and also petunia by Power et al. (1976) in England (Carlson et al., 1972; Power et al., 1976). Since then, regenerating plant protoplast has been reported for over 320 plant species including 146 plant genus and 49 plant families. The potential of using varieties obtained from somatic fusion has been examined in crops such as rice, rapeseed, tomato, potato, and citrus (Grosser et al., 2010). Generally, protoplast fusion is divided into two groups: spontaneous and inductive fusion. Protoplasts repel each other in inductive fusion due to negative charge around plasma membrane so that chemicals are used to reduce the electronegativity of protoplasts and consequently protoplasts can mix with each other (Verma et al., 2004). Some fusion methods include spontaneous fusion, fusion with PEG, fusion with calcium and high pH, fusion using nitrite sodium (NaNO5), and fusion with electric shock (Avratilova, 2004). Different cases may happen when two protoplasts fuse. A real hybrid is obtained if two nuclei fuse. If the two nuclei are separate and don’t fuse, the cell is called heterokaryon. Cells are called cybrid (cytoplasmic hybrid) if one of the nuclei is destroyed and only one remains in the mix of two cytoplasms (Avratilova, 2004).

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Saffron is a sterile plant and classical breeding methods are not applicable on it and hence protoplast fusion or induced mutation can be used for its breeding programs. Isa et al. (1990) isolated the protoplast from saffron for the first time. They used the enzymatic extraction method for protoplast isolation from calli derived from apical bud and corms. For this, cellulase R-10 from Trichoderma viridae and 1% driselase from Irpex lactes supplemented with low concentration of pectinase, pectolyase, and 0.3 M mannitol at pH 5.7 were used as digestion mixture at 25 C under dark conditions for 1
3 hours. Darvishi et al. (2007) also used MES in their enzyme mixture and the concentration of isolated protoplast was 40 3 105 protoplasts per ml with 98% viability. Protoplast immobilization in Ca-alginate, nurse culture, and plating density affected the growth and division of protoplasts (Isa et al., 1990). Cell division from protoplasts improved using the nurse culture. Also, the optimum density for higher frequency of colony formation was 5 3 104 protoplasts per mL. Isa et al. (1990) regenerated the shoots from calli of protoplasts. Noori-Daloii et al. (2000) used MS liquid medium containing pectolyase, cellulase, deriselase, and mannitol to separate saffron protoplast from callus. Isolated protoplasts were transferred to MS liquid medium containing different concentrations of hormones where seedling regeneration was performed. Hasrak and Zarghami (2013) used embryogenic calli to isolate saffron protoplast. Calli were placed on MS medium containing 0.1% pectolyase, 1% cellulase, 1% deriselase, 0.1% MES, and 0.3 M mannitol with temporal treatment of 1.5, 3, 4, and 5 hours. The results showed that 3 hours of enzymatic treatment of embryogenic calli of saffron was suitable for isolation and extraction of a high number of protoplasts with high viability. A study was performed to determine the best hormone compound for producing callus from protoplast and regenerating seedlings. Embryogenic calli were used to isolate protoplasts. The results showed that 1 mg L21 NAA and BAP treatments were the most suitable enzymatic treatment for seedling regeneration from calli of saffron (Chalooshi et al., 2006). Karamian and Ebrahimzadeh (2001) used the isolation method by Isa et al. (1990) for isolating protoplast from Crocus cancellatus. At first, embryogenic callus initiated from shoot meristems in medium supplemented by kinetin and 2,4-D was used for protoplast isolation. Then immobilization of protoplast in Ca-aliginate beads followed by nurse culture in a medium containing 4.4 μM 2,4-D, 8.9 μM kinetin and 0.57 mM ascorbic acid in dark conditions led to the highest growth and cell division. Langari-Sfezar (2017) isolated protoplasts from leaf mesophyll and flowers of saffron. They used cellulase, pectinase, mannitol, and MES as enzyme extraction mixture for isolating protoplast according to the Yoo et al. (2007) protocol and obtained 1.5 3 106 and 8.7 3 105 protoplasts per mL from leaf and flower petal, respectively. Also, the petal protoplasts were fused by 40% PEG.

14.8

Stigma-like structure

Tissue culture stigma or SLS refer to direct or indirect regeneration of stigmas through in vitro culture. The first report on SLS production was in 1987 by Sano and Himeno. They used young stigma plus ovaries, single stigmas, and half ovaries as explant for stigma production, from which half the ovaries were the best explant for SLS production with 75 SLS per explant. SLS also has crocin, picrocrocin, safranal, and crocetin metabolites, which are found in natural stigmas, but their contents in SLS are lower than natural stigmas (Sarma et al., 1991). Immature ovaries, half ovaries, stigmas, stigmas plus ovaries, anthers, stamens, and petals can produce SLS in special medium (Fakhrai and Evans, 1990; Namera et al., 1987; Sharma and Piqueras, 2010; Zhao et al., 2001). There are problems for proliferation of SLS in vitro culture due to low frequency of generation, formation of non-SLS structures, browning of SLS, slow growth of callus, and single harvest. To overcome these challenges, different attempts have been made. For example, Loskutov et al. (1999) used B5 medium, subculturing at short intervals, and activated charcoal for harvesting SLS within a period of 10 months. Moreover, casein hydrolysate and L-alanine improved the induction of SLS and concentration of crocin, crocetin, safranal, and picrocrocin to levels higher than the concentrations of natural stigmas (Zeng et al., 2003). Using precursors of coloring metabolite, it is possible to increase their contents in SLS. L-Alanine and sodium carbonate can increase the synthesis of Acetyl CoA in plant and Acetyl CoA used for terpenoid production such as crocin and crocetin (Otsuka et al., 1992; Zeng et al., 2003). The amount of crocin in SLS is also affected by light (4.91% on medium with light and 2.21% in dark) (Zeng et al., 2003). Direct and indirect induction of SLS depends on growth regulators and their combination. Low concentrations of NAA and BA induce direct generation, while high concentrations induce indirect SLS generation. Also, the metabolites of direct SLS are more comparable with natural stigmas (Ebrahimzadeh and Karamian, 2000; Loskutov et al., 1999). There is no report on the optimum temperature for SLS induction, however, considering that saffron flowers in the cold season low temperatures between 20 C and 25 C seem to be suitable for SLS production. In some studies, the temperature for SLS induction was 25 C or room temperature (Loskutov et al., 1999; Zeng et al., 2003).

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Mir et al. (2010) regenerated the SLS from half ovary explants both directly and indirectly. They used B5 medium supplemented by 27 μM NAA and 44.4 μM BA for developing SLS with 5.2 cm size, but their number using the indirect method (120) was more than the direct method (20) (Fig. 14.6).

FIGURE 14.6 (A) SLS production in saffron, (B) indirect SLS development from half ovary, and (C) direct SLS development from half ovary. From Mir, J.I., Ahmed, N., Wani, S.H., Rashid, R., Mir, H., Sheikh, M.A., 2010. In vitro development of microcorms and stigma like structures in saffron (Crocus sativus L.). Physiol. Mol. Biol. Plants 16 (4), 369
373.

TABLE 14.1 Summary of in vitro research on saffron. Explant/callus

Results

References

Corm

Callus, shoots

Ding et al. (1981)

Corm

Callus, shoots

Ilahi et al. (1987)

Corm

Direct shoot regeneration

Homes et al. (1987)

Ovary, stigma

Stigma-like structure formation

Sano and Himeno (1987)

Apical bud

Direct shoot regeneration, micricorm formation

Plessner et al. (1990)

Callus from corms

Protoplast, shoot regeneration

Isa et al. (1990)

Anthers, ovary

Stigma-like structure formation

Sarma et al. (1991)

Apical buds

Callus, somatic embryogenesis

George et al. (1992)

Corm

Somatic embryogenesis

Ahuja et al. (1994)

Ovary

Callus, shoots

Igarashi and Yuasa (1994)

Corm, shoot, inflorescence

Callus, somatic embryogenesis

Milyaeva et al. (1995)

Ovary

Callus, stigma-like structure formation

Castellar and Iborra (1997)

Apical bud

Callus, somatic embryogenesis, microcorm

Piqueras et al. (1999)

Callus

Protoplast culture

Noori-Daloii et al. (2000)

Style, perianth

Stigma-like structure formation

Ebrahimzadeh and Karamian (2000)

Petal, stigma, style, corm

Callus, stigma-like structure formation

Zeng et al. (2003)

Corm, callus

Crocin synthesis in cell cultures

Chen et al. (2003)

Corm

Crocin synthesis in cell cultures

Chen et al. (2004)

Apical bud

Callus, somatic embryogenesis, protoplast

Darvishi et al. (2007)

Corm, shoots, inflorescence, ovary, flower, stigma

Shoot, somatic embryogenesis, microcorm

Karaoglu et al. (2007) (Continued )

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TABLE 14.1 (Continued) Explant/callus

Results

References

Leaf, somatic embryos

Callus, somatic embryogenesis, shoots, microcorm formation

Raja et al. (2007)

Somatic embryos

Microcorm formation

Sheibani et al. (2007)

Apical bud

Shoot induction, plantlet regeneration

Sharifi et al. (2010)

Embryogenic calli

Protoplast culture

Hasrak and Zarghami (2013)

Corm slices

Cormlet induction

Parray et al. (2012)

Corms

Direct shoot formation

Zeybek et al. (2012)

Lateral and apical buds

Adventitious shoots (direct and indirect)

Simona et al. (2013)

Corm

Adventitious shoots

Morata et al. (2013)

Apical shoots

Microcorm

Mir et al. (2014)

Corm segments

Microcorm

Hagizade (2016)

Leaf mesophyll and flowers

Protoplast isolation

Langari-Sfezar (2017)

Apical and lateral buds

Microcorm formation

Sargazi-Moghadam (2019)

14.9

Conclusion

Saffron is a sterile triploid plant that is only reproduced through vegetative methods, but the low rate of classical propagation is a limiting factor for expanding saffron cultivation areas. Therefore, in vitro propagation is important for development of this economic plant. In vitro saffron corm production has led to effective in vitro mass production of uniform and disease-free cormlets. Contaminated corms cause decrease or even stop development and flowering. In addition, uniform-size corms with the aim of better flowering are obtained in tissue culture. More importantly, different genetic variation induction methods can be utilized as saffron modification methods if in vitro and regeneration culture methods are optimized. Different attempts have been made for micropropagation of saffron. Somatic embryogenesis, indirect organogenesis, and SLS generation are ready for large-scale production, but more cost-effective methods need to be developed. Currently direct shoot formation and cormogenesis are promising ways for commercial propagation of saffron in the short term (Agayev et al., 2009). However, three concerns should be considered: (1) sustained multiplication rate of shoots, (2) developing large-sized cormlets, and (3) agronomic performance of microcorms. The latter is very important, and there is only one report on the subject (Parray et al., 2012). Moreover the studies on saffron show that different factors affect shoot multiplication and cormogenesis such as light, temperature, growth regulators, and their interactions. In saffron, BA is a signaling hormone for cormogenesis and antigibberellic agents promote corm induction. For commercial micropropagation of saffron, the cost should be decreased specially for the high cost related to labor, agar, and sucrose, which are consumed in vitro cultures. Using SLS cultures and cell culture of saffron for metabolite production are alternative methods for natural stigmas. But the amounts of crocin, crocetin, safranal, and picrocrocin in SLS and cell culture are lower than that in the natural stigmas. Supplementation of culture media with some precursors such as L-alanin seems to improve the crocin and crocetin content in SLS. For commercial production of saffron metabolites in bioreactors, factors like media, casein hydrolysate, precursors, heavy or rare metals, temperature, light, and a two-stage culture system should be optimized (Sharma and Piqueras, 2010). Some in vitro research on saffron is summarized in Table 14.1.

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16. Schenk, R.U., Hildebrandt, A.C., 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50 (1), 199
204. Sharafzadeh, Sh, 2012. In vitro callus induction saffron leaves. Int. J. Pharma Bio Sci. 3 (1), 171
175. Sharifi, G., Ebrahimzadeh, H., Ghareyazie, B., Karimi, M., 2010. Globular embryo-like structures and highly efficient thidiazuron-induced multiple shoot formation in saffron (Crocus sativus L.). In Vitro Cell. Dev. Biol. Plant 46, 274
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24. Sharma, K.D., Rathour, R., Sharma, R., Goel, S., Sharma, T.R., Sing, B.M., 2005. Development of low cost media for in vitro shoot regeneration in saffron (Crocus sativus). Indian Perfumer 49, 333
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6 September 2012, Mashhad, (in Persian). Vasil, V., Vasil, I.K., 1979. Isolation and culture of cereal protoplasts I. Callus formation from pearl millet (Pennisetum americanum) protoplasts. Z. Pflanzenphysiol. 92, 379
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6 September 2012, Mashhad (in Persian).

Chapter 15

Molecular biology of Crocus sativus Alireza Seifi and Hajar Shayesteh Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 15.1 15.2 15.3 15.4

Introduction Flower development Gametogenesis and interspecific hybridization Secondary metabolites 15.4.1 Carotenoid biosynthesis in plants 15.4.2 Carotenoid biosynthesis in saffron 15.4.3 Apocarotenoid biosynthesis

15.1

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15.4.4 Crocin, crocetin, picrocrocin, and safranal 15.4.5 Genetic regulation of carotenoids biosynthesis 15.5 Production of saffron metabolites in microorganisms 15.6 Saffron
microbe interactions 15.7 Molecular response to abiotic stresses 15.8 Conclusion References

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Introduction

Saffron (Crocus sativus) was once an orphan crop for plant molecular biologists and geneticists and only limited molecular information was available. Fortunately, the trend has changed significantly and many researchers in different parts of the world including Europe, India, Iran, Turkey, and Pakistan are now engaged in molecular studies of saffron. Here we summarize advances in saffron molecular genetics and biology that have deepened our understanding of development, secondary metabolism, interaction with microorganisms, and response to environmental stresses of this valuable crop.

15.2

Flower development

Flowers, and more specifically the stigmata, are the commercially valuable organs in saffron, and thus flower development in saffron has turned into an exciting and fast moving field of research. Flower organs are organized in four whorls, namely sepals, petals, stamens, and carpels. A group of homeotic transcription factors called MADS-box proteins, composed of different classes A, B, C, D, and E, are the major players in flower organ identity and development (Prunet and Meyerowitz, 2016; Theißen et al., 2016). Based on the current model of flower development, known as the Quartet model, the first whorl (sepals) is determined by class A and E genes, the second whorl (petals) is specified by class B and E genes, the third whorl (stamens) is determined by the class B, C, and E genes, the fourth whorl (carpels) is determined by class C and E genes, and ovule development in the carpels is under control of the class C, D, and E genes (Theißen et al., 2016). The genes identified in saffron that are involved in flower development are summarized in Table 15.1. Tsaftaris and coworkers cloned the first MADS-box genes from saffron. They cloned and characterized three homologous APETALAlike genes from C. sativus: CsAP1-a, CsAP1-b, and CsAP1-c (Tsaftaris et al., 2004). Later, two AGAMOUS1-like genes (CsAG1) were identified (Tsaftaris et al., 2005). These two genes, which are two isoforms resulting from alternative splicing, are expressed in flowers particularly in stamen and carpels (Tsaftaris et al., 2005). Five PISTILLATA/ GLOBOSA-like MADS-box genes have been identified in saffron. Interestingly the expression pattern of the genes is different from their expression in Arabidopsis; they are not only expressed in the second and third whorls but also in the first whorl (Kalivas et al., 2007). Three SEPALIATA3 (SEP3)-like genes that are identified in saffron are expressed in all flower organs but not in the leaves (Tsaftaris et al., 2011). The orthologues of APETALA2 gene in saffron, CsAP2, is expressed in all the tested organs (Tsaftaris et al., 2012b). Ectopic expression of CsatCEN/TFL1-like, Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00015-0 © 2020 Elsevier Inc. All rights reserved.

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TABLE 15.1 Genes involved in flower development in Crocus Sativus. Genes in C. sativus

Orthologues in Arabidopsis

MADS-box class

Organ of expression

Reference

CsAP1

APETALA1

A

Sepals and petals

Tsaftaris et al. (2004)

CsAP2

APETALA2




Corm, leaf, flower

Tsaftaris et al. (2012b)

CsAP3

APETALA3

B

Petal and stamen

Wafai et al. (2015)

CsPI

PISTALLATA

B

Petal and stamen

Kalivas et al. (2007)

CsAG

AGAMOUS

C

Stamens and carpels

Tsaftaris et al. (2005)

CsMYB1

MYB




Stigma

Go´mez-Go´mez et al. (2012)

CsSEP3

SEPALIATA3

E

Flower

Tsaftaris et al. (2011)

a CENTRORADIALIS/TERMINAL FLOWER1 (CEN/TFL1)-like gene, in Arabidopsis tfl1 mutant restored the wild phenotype (Tsaftaris et al., 2012a). CsMYB1, an R2R3 MYB factor from saffron, was identified that interestingly shows no expression in those Crocus species with branched stigma morphology, indicating that this transcription factor is probably involved in stigma morphology (Go´mez-Go´mez et al., 2012). Flower development is not a very tuned process; on the contrary, like any other biological processes, flowering is a regulated process but with a level of stochasticity. For example, with a very low frequency, about 1.2 3 1026, there are saffron flowers with abnormal numbers of flower organs, like stigma numbers more than three (Estilai, 1978; Ghaffari and Bagheri, 2009). Cytological and morphological examinations have revealed that this abnormality is not controlled genetically, but it is probably because of fusion of two or more buds (Ghaffari and Bagheri, 2009). In fact, variation in floral organ is a common phenomenon in many other plant species, and is mainly the result of stochasticity in organ fate determination during flower development (Kitazawa and Fujimoto, 2014).

15.3

Gametogenesis and interspecific hybridization

In angiosperms, male and female gametes are produced during microsporogenesis and megasporogenesis, respectively. In anthers, microsporocytes go through meiotic divisions to produce microspores, which will form male gametophyte (pollen grain) by subsequent mitotic divisions. In an analogous scenario in ovules, megasporocytes produce magaspores by meiosis, which eventually form the female gametophyte (embryo sac). The key to successful gametogenesis is to have normal meiosis divisions. In diploid species, meiosis is a straightforward phenomenon and often results in haploid gametes. But for triploid cells the chance to get haploid gametes with a whole set of chromosomes is very low, and therefore triploids are sterile. Although pollen grains of saffron are mostly alive (65% based on nitroblue tetrazolium test), they have a very low germination rate that can be justified by the cytological abnormalities (Caiola, 2005; Chichiricco and Caiola, 1986). The pollen tube cannot penetrate the ovule in C. sativus, suggesting that C. sativus probably originated from a self-incompatible species (Chichiricco and Caiola, 1986). This self-incompatibility lessens the likelihood of seed production in saffron close to impossible. Saffron seeds can be produced by interspecific hybridization with diploid Crocus species (Caiola and Canini, 2010). Hand pollination of C. sativus flowers with pollens from Crocus cartwrightianus resulted in formation of seeds with different color and larger dimension compared with the diploid seeds. The seeds germinated with high percentage similar to diploid seeds, and resulted in seedlings, which subsequently produced corms without tonics. Although the corms produced cormlets after 3 years, the flowering stage of the corms was not observed eventually (Maria Grili Caiola, personal communication, September 29, 2017).

15.4

Secondary metabolites

Plant secondary metabolites are those compounds that, unlike primary metabolites (e.g., carbohydrates, lipids, and proteins), are not necessarily required for normal growth and development, but they are mainly important for plant interactions with the environment. Based on their biosynthetic origin, secondary metabolites are classified into five major groups: terpenoids, alkaloids, cyanogenic glucosides, glucosinolates, and phenolic compounds (Buchanan and Jones, 2007).

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Terpenoids, also known as isoprenoids or terpenes, are all composed of basic five-carbon isopentate (isoprene) units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are isomers synthesized in the mevalonate pathway (MEV) in cytosol, or the nonmevalonate methylerythritol 4-phosphate pathway (MEP/DOXP) in plastids (Kirby and Keasling, 2008). Alkaloids are usually synthesized from common amino acids and function in chemical defense against herbivores (Croteau et al., 2000). Cyanogenic glucosides are characterized by their ability to release hydrogen cyanide (cyanogenesis) upon hydrolysis by beta-glycosidases. This makes them essential defense compounds against herbivores. Glucosinolates, which are mainly restricted to Brassicales, are sulfur-rich anionic betathioglycosides derived from amino acids. Phenolic compounds, which possess one or more hydroxyl groups attached to an aromatic arene (phenyl) ring, are primarily synthesized in phenylpropanoid and phenylpropanoid acetate pathways, with phenylalanine as the precursor (Buchanan and Jones, 2007). Their structures may range from simple molecules (phenolic acids) to polyphenols and polymeric compounds. Anthocyanins, which are responsible for most of the red, purple, and blue colors in flowers and fruits, belong to a large family of polyphenolic plant compounds known as flavonoids. They have important roles in attraction of pollinators and seed dispensers (Cheynier, 2012; Croteau et al., 2000).

15.4.1 Carotenoid biosynthesis in plants Carotenoids are tetraterpenoids (C40) that are derived from two isoprene isomers, IPP and DMAPP. Although there are two biosynthetic pathways for building these C5 units, the cytosolic MEV and the plastidic MEP/DOXP pathway, only the latter is mainly involved in carotenoid biosynthesis in plants (Eisenreich et al., 2004; Rodrıguez-Concepcio´n and Boronat, 2002). In MEP pathway, deoxyxylulose-5-phosphate (DXP) is formed from pyruvate and glyceraldehyde-3phosphate by the action of DXP synthase (Fig. 15.1). The next step is carried out by 1-deoxyxylulose-5-phosphate

FIGURE 15.1 Schematic diagram of the carotenoid biosynthesis pathways in plants. In MEP pathway, condensation of G3P and pyruvate catalyzed by DXS to produce DXP, which is then reduced by DXR to form MEP. After a series of catalytic reactions, IPP and DMAPP are formed, which then join together by GGPPS to produce GGPP. PSY catalyzes the first step in the carotenoid specific pathway that converts two GGPP to cis
phytoene. The action of desaturase and isomerase enzymes result in formation of lycopene. This is then cyclized by β-LCY and ε-LCY or β-LCY to form α-carotene and β-carotene. α-Carotene is twice hydroxylated by ε- and β-OHases to form lutein. Two β-ring hydroxylations of β-carotene by β-OHase give rise to zeaxanthin. Finally, different volatiles (e.g., β-citraurin) and phytohormones (strigolactone and abscisic acid) are produced by CCDs and NCEDs: β-LCY, β-cyclase; β-OHase, β-carotene hydroxylase; CCD, carotenoid cleavage dioxygenase; CRTISO, carotenoid isomerase; DMAP, dimethylallyl diphosphate; DXP, deoxy-D-xylulose 5-phosphate; DXS, DXP synthase; DXR, DXP reductoisomerase; ε-LCY, ε-cyclase; ε-OHase, ε-carotene hydroxylase; G3P, glyceraldehyde-3-phosphate, GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase, IPP, isopentenyl diphosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; NCED, 9-cisepoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; PSY, phytoene synthase; ZDS, ζ-carotene desaturase; ZEP, zeaxanthin epoxidase; and Z-ISO, ζ-carotene isomerase. From Nisar, N., Li, L., Lu, S., Khin, N.C., Pogson, B.J., 2015. Carotenoid metabolism in plants. Mol. Plant 8, 68
82.

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reductoisomerase (DXR), which converts DXP to MEP. After a series of catalytic reactions, IPP and DMAPP are formed. The combination of these molecules by prenyltransferases leads to the production of geranyl diphosphate (GPP), farsenyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP), which are the precursors of monoterpenoids (C10), sesquiterpenoids (C15), and diterpenoids (C20), respectively (Kirby and Keasling, 2008). Carotenoid biosynthesis starts with the condensation of two GGPP molecules by phytoene synthase (PSY) giving rise to C40 linear 15-cis-phytoene. Phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) catalyze desaturation reactions converting 15-cis-phytoene to tetra-cis-lycopene (Fig. 15.2). Two specific isomerase enzymes, carotenoid isomerase (CRTISO) and ζ-carotene isomerase (Z-ISO), convert poly-cis compounds to their all-trans isomers. Thus, all-trans lycopene, the first pigmented carotenoid, is formed through a series of desaturation and isomerization. Lycopene is cyclized by two competing enzymes, lycopene β-cyclase (β-LCY) and lycopene Ɛ-cyclase (Ɛ-LCY). On one hand, the addition of one and two β-rings to lycopene by β-LCY produces ɤ-carotene and orange pigment β-carotene, respectively. On the other hand, the addition of ε- and then β-rings to lycopene by Ɛ-LCY and β-LCY leads to formation of δ-carotene and α-carotene, respectively (Nisar et al., 2015). Hydroxylation of α-carotene in two sequential steps by heme-containing cytochrome P450 monooxygenases (CYP97A, CYP97C) yield to lutein, while two β-ring hydroxylations of β-carotene by β-carotene hydroxylase (BCH) give rise to zeaxanthin (Sandmann et al., 2006).

FIGURE 15.2 Schematic representation of carotenoid and apocarotenoid biosynthesis pathways in Crocus sativus. Carotenoid biosynthesis starts with the condensation of two GGPP molecules by PSY giving rise to phytoene. This is converted into lycopene in a series of desaturation and isomerization reactions, which are catalyzed by PDS and ZCD (desaturases), Z-ISO and CRTISO (isomerases). Lycopene is cyclized by β-LCY and ε-LCY or β-LCY to form α-carotene and β-carotene. The cleavage of β-carotene furcated by CCD1, CCD4, CCD7, and CCD8 yield to β-ionone, C14 dialdehyde, hydroxy-β-ionone, β-cyclocitral, and strigolactone. Moreover, hydroxylation of β-carotene and α-carotene give rise to zeaxanthin and lutein, respectively. Zeaxanthin is cleaved by CCD2 to produce crocetin dialdehyde and hydroxy-β-cyclocitral. Crocetin dialdehyde is further dehydrogenated and glycosylated to crocetin and crocin by ALDH and UGT, respectively. Hydroxy-β-cyclocitral is converted to picrocrocin by an UGT, and then to safranal. In the other branch point, zeaxanthin is converted into violaxanthin and neoxanthin by ZEP and NSX, respectively. Neoxanthin is cleaved by NCED to produce xanthoxin, precursor of abscisic acid (ABA). ALDH, aldehyde dehydrogenase; β-LCY, β-cyclase; β-OHase, β-carotene hydroxylase; CCD, carotenoid cleavage dioxygenase; CRTISO, carotenoid isomerase; ε-LCY, ε-cyclase; ε-OHase, ε-carotene hydroxylase, NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; PSY, phytoene synthase; UGTs, UDPG-glucosyltransferase; ZDS, ζ-carotene desaturase; ZEP, zeaxanthin epoxidase; Z-ISO, ζ-carotene isomerase. Modified from Baba, S.A., Ashraf, N., 2016. Apocarotenoids of Crocus sativus L: From Biosynthesis to Pharmacology. Springer, Singapore.

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15.4.2 Carotenoid biosynthesis in saffron Saffron has many volatile and nonvolatile active compounds belonging to different groups of secondary metabolites, including carotenoids, monoterpenoids, and flavonoids. Carotenoids, which are the most important pigments in saffron stigmas, are mainly C40 isoprenoids including α- and β-carotene and zeaxanthin (Gresta et al., 2008). C20 apocarotenoids such as crocetin and its ester derivatives are produced from the oxidative cleavage of carotenoids. Crocin is the glycosylated form of crocetin, both of which are responsible for the red color of saffron (Carmona et al., 2006). The taste and aroma of saffron are mainly due to the accumulation of picrocrocin and safranal, respectively. Picrocrocin is a colorless glycoside precursor of safranal and is formed from the enzymatic degradation of zeaxanthin (Srivastava et al., 2010). The cDNAs encoding PSY and PDS are cloned in saffron (ScPYS and CsPDS), and expression analysis showed that the highest level of their expression occurs in the orange stage of the stigma (Castillo et al., 2005). Two lycopene cyclases have been identified in saffron (CsLCYB1 and CsLCYB2a). The CsLcyB2a gene is absent in Crocus kotschyanus, while it is present in Crocus goulimyi and Crocus cancellatus (Ahrazem et al., 2009). Two saffron genes encoding BCH, CsBCH1, CsBCH2, have been identified (Castillo et al., 2005). An ortholog of the CRTISO has been identified in saffron, but it needs further characterization (Baba et al., 2015b). Both β-carotene and zeaxanthin are substrates for the biosynthesis of unique apocarotenoids in saffron.

15.4.3 Apocarotenoid biosynthesis The catalytic activity of carotenoid cleavage dioxygenases (CCDs) along with enzymatic oxidation via peroxidases/ lipoxygenases or nonenzymatic photochemical oxidation in photosynthetic tissues under high light stress are responsible for formation of various apocarotenoid compounds, which have diverse roles in plants as phytohormones, signal molecules, and volatile/flavor compounds (Auldridge et al., 2006; Nisar et al., 2015). According to their substrate preference and/or the cleavage position, plant carotenoid cleavage oxygenases are grouped into two big families: 9-cis-epoxycarotenoid dioxygenases (NCEDs) and CCDs (Auldridge et al., 2006; Walter and Strack, 2011). An ortholog of the NCED gene was identified in saffron. The expression of CsNCED in floral and corm tissues correlated with ABA levels, suggesting a possible role of the gene in regulation of stigma senescence and corm dormancy in C. sativus (Ahrazem et al., 2011). Two isoforms of CCD1 have been isolated from C. sativus (CsCCD1a and CCD1b) that cleave zeaxanthin and β-carotene at the 9, 10 double bond to produce C14 dialdehyde and β-ionone, respectively. CsCCD1a is ubiquitously expressed while CsCCD1b expression is specific to stigma (Rubio et al., 2008). Except for CCD1, which is located in cytosol, other CCDs have a plastid-targeting transit peptide and act in plastids (Rubio et al., 2008; Ahrazem et al., 2016). Through transcriptome sequencing CsCCD2 was identified and characterized (Frusciante et al., 2014). The gene is expressed early during stigma development and is able to cleave zeaxanthin at sequentially the 7, 8 and 70 , 80 double bonds adjacent to a 3-OH β-ionone ring, converting it to crocetin dialdehyde. CsCCD2 is closely related to CsCCD1 (with 97% identity) and its preferred substrates are lutein and zeaxanthin, but not β-carotene, as shown by in vitro cleavage assay. According to the sequence of CsCCD2 gene, the authors suggested that this gene lacks a plastid transit peptide, and thus is localized in the chromoplast outer envelope (Frusciante et al., 2014). However, another research group characterized CaCCD2 from Crocus ancyrensis and compared its sequence with CsCCD2. There was a clear length difference between these two genes at the 50 end, suggesting that the sequence of CsCCD2 was not reported correctly originally. The 50 -RACE PCR analysis of CsCCD2 and cloning the longest cDNA isolated from C. sativus stigma revealed that the full sequence, named CsCCD2L, encodes a protein, which is 60 amino acids longer than the CsCCD2, with a plastid transit peptide (Ahrazem et al., 2016) and localized in plastids (Demurtas et al., 2018). Further analysis resulted in identification of three CsCCD2 paralogs in C. sativus. The longest gene, CsCCD2a, consisted of nine introns and ten exons. The CsCCD2b has nine exons and eight introns. The shortest one, CsCCD2-t, is a truncated gene. RNA-seq studies in three developmental stages of saffron stigma revealed that intron retention is the common form of alternative splicing in CsCCD2 (Ahrazem et al., 2016). The CCD4 family is the largest family of plant CCDs and plays a significant role in the level of organ pigmentation including citrus peel (Rodrigo et al., 2013), Arabidopsis seeds (Gonzalez-Jorge et al., 2013), and potato tubers (Campbell et al., 2010), and in volatile emission during flowering in saffron (Rubio et al., 2008). Rubio et al. (2008) showed that expression patterns of CsCCD4a and CsCCD4b are consistent with the highest levels of β-carotene and emission of β-ionone during the stigma development. This volatile compound plays a role in attracting insect pollinator especially in those Crocus species that are self-incompatible and have heavy pollen grains to transfer with air

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(Rubio et al., 2008). A new member of the CCD4 family from C. sativus, named CsCCD4c, was isolated later (Frusciante et al., 2014). The expression of this intronless gene is restricted to stigmas and induced by heat, osmotic stress, and wounding, suggesting the role of its products in the adaptation of saffron to environmental stresses (Frusciante et al., 2014). The expression level of CsCCD4a and CsCCD4b genes increases in response to dehydration, salt, and methylviologen (Baba et al., 2015a). Transgenic Arabidopsis plants overexpressing CsCCD4b developed longer roots with a higher number of lateral roots and displayed higher activity and expression of reactive oxygen species (ROS) scavenging enzymes. These results indicate that β-ionone and β-cyclocitral, products of CsCCD4b, may be involved in plant responses to dehydration, salinity, and oxidative stresses (Baba et al., 2015a). Some CCD4 enzymes in other plants, like VcCCD4a and VcCCD4b in Vitis vinifera, cleave carotenoids at 5, 6 (50 , 0 6 ) double bonds (Lashbrooke et al., 2013), or like citrus CCD4 at 70 , 80 double bonds (Gonzalez-Jorge et al., 2013; Rodrigo et al., 2013). In saffron, zeaxanthin cleavage dioxygenase (CsZCD) was first reported to cleave zeaxanthin at the 7, 8/70 , 80 positions to produce crocetin dialdehyde (Bouvier et al., 2003). However, sequence comparison and structure prediction revealed that CsZCD is an N-truncated form of CsCCD4, missing one blade of the β-propeller structure, and therefore does not have cleavage activity (Rubio et al., 2008). CCD7 works in sequence with CCD8 to synthesize strigolactone, an apocarotenoid hormone that inhibits plant shoot branching. First, β-carotene is cleaved by CCD7 to produce 100 -apo-β-carotenal and β-ionone. Then, the cleavage of 100 -apo-β-carotenal by CCD8 leads to formation of C18-ketone β-apo-13-carotenone that, via several different reactions, is converted to strigolactone precursor carlactone (Seto et al., 2014). These molecules also stimulate the growth of symbiotic mycorrhizal fungi (Akiyama et al., 2005) and the germination of parasitic plant seeds (Cook et al., 1972). In C. sativus, CsCCD8 is highly expressed in quiescent axillary buds, and its expression is significantly decreased by decapitation indicating its involvement in axillary bud outgrowth. In addition, CsCCD8 may have a significant function in the control of apical dominance. The abundance of CCD7 and CCD8 in immature orange stigmas and reduction of both genes in the senescent stigma suggests an interesting novel function for the enzymes and of strigolactones (Frusciante et al., 2014).

15.4.4 Crocin, crocetin, picrocrocin, and safranal As described above, production of bioactive compounds in saffron engages the MEP pathway from pyruvate and glyceraldehyde-3-phosphate to GGPP, the carotenoid pathway from GGPP to zeaxanthin, and the crocin pathway from zeaxanthin to crocin. In the latter one, symmetric cleavage of zeaxanthin at the 7, 8/70 , 8 positions by CsCCD2 yields to crocetin dialdehyde and 3-OH-β-cyclocitral. On one hand, crocetin dialdehyde is further dehydrogenated and glycosylated to crocetin and crocin by an aldehyde dehydrogenase (ALDH) and UDPG-glucosyltransferase (UGT), respectively. On the other hand, hydroxy-β-cyclocitral is converted to picrocrocin by an UGT, and then to safranal (Frusciante et al., 2014). Five different ALDHs were identified in saffron; expression of two of them (ADH2946 and ADH11367) had a high correlation with crocetin concentration, and showed the highest expression level at the anthesis stage. Also, there was a third candidate (ADH54788) with an expression pattern like crocetin (Go´mez-Go´mez et al., 2017). The ADLH enzymes are localized in the endoplasmic reticulum (Demurtas et al., 2018). Crocin is the most important metabolite of saffron stigma and quickly dissolves in water. Besides the Crocus genus, crocin has also been identified in other plants such as Buddleja officinalis, Nyctanthes arbor-tristis, and Gardenia jasminoides (Gadgoli and Shelke, 2010; Liao et al., 1999; Pfister et al., 1996). Depending on the number of sugar molecules, different forms of crocin are produced. Different forms of crocin including crocetin β-D-glucosyl ester, crocetin β-D-gentiobiosyl ester, di-(β-D-glucosyl) ester, crocetin β-D-gentiobiosyl-β-D-glucosyl ester, and crocetin di-(β-D-gentiobiosyl) ester are discernable in HPLC analysis (Moraga et al., 2004). The crocetin di-(β-D-gentiobiosyl) ester (α-crocin) is the most abundant form of crocin in saffron and because of high water solubility it has the maximum coloring capacity, making it a good candidate for applications in foods as colorant (Tarantilis et al., 1995). β-Crocetin (mono methyl ester of crocetin) and γ-crocetin (dimethyl ester of crocetin) are minor components among water-soluble C20 apocarotenoids of saffron (Ferna´ndez, 2004). Moraga et al. (2004) reported the cloning of two GTase genes from C. sativus. CsUGT2 was expressed only in stigma of Crocus species that synthesize crocin and the expression pattern was consistent with accumulation of crocetin with higher sugar moieties. Transcripts of CsUGT3 and other structural genes for carotenoid biosynthesis were detected in the stigma tissue of all tested Crocus species. Expression of CsUGT2 in Escherichia coli demonstrated the glucosylation activity of recombinant protein against crocetin, crocetin β-D-glucosyl ester, and crocetin β-D-gentibiosyl ester (Moraga et al., 2004). Interestingly, CsUGT2 is expressed in stigma-like structures (Namin et al., 2009). In C.

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ancyrensis, high expression of an ortholog of CsUGT2 was correlated with high levels of crocin accumulation in stigma and tepals (Ahrazem et al., 2015). There is no UGT identified yet for conversion of hydroxy-β-cyclocitral to picrocrocin, a monoterpene glycoside, which is considered the main bitter taste of saffron. Hydrolysis of picrocrocin gives rise to safranal, the main constituent of the volatile oil fraction (30%
70% of the essential oil). Besides safranal, other aromatic compounds with the same skeleton such as isophorone, 2,2,6-trimethyl-1,4-cyclohexanedione, 4-ketoisophorone, 2-hydroxy-4,4,6-trimethyl-2,5cyclohexadien-1-one, and 2,6,6-trimethyl-1,4-cyclohexadiene-1-carboxaldehyde add to the aroma of the spice and seem to be derived from picrocrocin (Maggi et al., 2009). However, identification of several new glycosides indicates their role as the glycosidic aroma precursor along with picrocrocin (Carmona et al., 2006). Although studies show that dehydration is important for production of safranal (Himeno and Sano, 1987; Raina et al., 1996), it is not exactly clear yet whether this reaction happens enzymatically or nonenzymatically.

15.4.5 Genetic regulation of carotenoids biosynthesis The steady-state level of carotenoids is dependent on the rate of their biosynthesis, degradation, and storage capacity of the cell. Plants have multiple mechanisms controlling production and accumulation of carotenoids including regulation of the expression of key carotenoid biosynthesis pathway genes and CCDs. During the development of saffron, the color of stigma changes from white to scarlet, passing through yellow, orange, and red stages, accompanying carotenoid accumulation. For example, in one study β-carotene and zeaxanthin reached 60.5% and 85%, respectively, in the scarlet stage (Castillo et al., 2005). Phytoene biosynthesis is a bottleneck in the carotenoid pathway. PSY gene is induced in response to different factors including temperature, drought, salt, high light, ABA, development, photoperiod, and posttranscriptional feedback regulation (Cazzonelli and Pogson, 2010). In Arabidopsis, RAP2.2 transcription factor binds to the promotion of PSY and PDS and overexpression of RAP2.2 results in reduction of PSY and PDS transcription and carotenoid levels (Welsch et al., 2007). Another transcription factor in Arabidopsis named RIF was also shown to bind to the PSY promoter under dark conditions and repress its expression (Toledo-Ortiz et al., 2010). In C. sativus, the lowest level of PSY and PDS transcripts, which is at the early developmental stages the abundance of PSY and PDS transcripts is low and then reaches to the highest level in the orange stage of the stigma, and then remained relatively constant through development (Castillo et al., 2005). The cyclization of lycopene is the other regulatory node in carotenoid biosynthesis pathway that is catalyzed by two lycopene cyclases, Ɛ-LCY and β-LCY. In Arabidopsis, silencing of Ɛ-LCY by cosuppression results in alteration of the ratios of lutein to β-carotene (Pogson et al., 1996). Two genes encoding lycopene β-cyclase are characterized in saffron: CsLcyB2a and CsLcyB2a (Ahrazem et al., 2009). The stable expression of CsLcyB2a in transgenic Arabidopsis showed lycopene β-cyclase activity and caused an increase of β-carotene. CsLcyB2a is expressed only in stigma tissue, where β-carotene accumulated, with the highest levels of expression in the days before anthesis. However, CsLcyB1 transcript is present in leaves, tepals, and stigmas at lower levels (Ahrazem et al., 2009). BCHs are key enzymes in the accumulation of apocarotenoids in C. sativus as they are directly involved in production of zeaxanthin as precursor of saffron apocarotenoids (Bouvier et al., 2003). Detection of BCH transcripts by RTPCR in fully developed stigma at the anthesis stage showed that massive accumulation of both CsBCH1 and CsBCH2 transcripts correlated with zeaxanthin and the accumulation of its derivations in saffron stigma (Castillo et al., 2005). Moreover, the abundance of CsBCH1 transcripts in different Crocus species was related to the their zeaxanthin content, suggesting that the reaction catalyzed by CsBCH1 enzyme could be the limiting step in the production of apocarotenoids in the saffron stigma (Castillo et al., 2005). The expression of the carotenogenic genes including PSY, ZDS-V, BCH, and LCY-II was correlated with accumulation of crocins in C. ancyrensis (Ahrazem et al., 2015). There are cis-regulatory motifs in the CCD2 promoter responding to light, temperature, and circadian regulation that cause higher expression levels during the night and under low temperatures (Ahrazem et al., 2016). Similar behavior was observed for the chromoplast-specific carotenogenic genes, CsLycB2a and CsBCH1, suggesting coregulation of these genes during the development of the stigma in saffron. In addition, the accumulation pattern of cyclocitral was correlated with the expression level of CsCCD2 and thus it could be assumed as a signal molecule from chromoplast to nucleus, which coordinates the expression of CsCCD2 with the developmental state of the chromoplast during stigma growth (Ahrazem et al., 2009, 2016). The maximum expression of CCD2 is at the orange stage, coincident with accumulation of crocetin and crocin (Frusciante et al., 2014). For the first time, D’Agostino and coworkers developed an expressed sequence tag (EST) collection from saffron mature stigma comprising 6603 high-quality ESTs, which facilitated identification of key genes involved in

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apocarotenoid metabolism and regulation (D’Agostino et al., 2007). Expression profiling from five different tissues/ organs of C. sativus revealed that key enzymes involved in apocarotenoid biosynthesis including CCD, glucosyltransferases, ALDHs and beta glucosidases expressed higher in stigma compare to other tissues, suggesting that apocarotenoids are produced mainly in stigma (Jain et al., 2016). Similar expression patterns were reported earlier for PSY, PDS, and CCD2 (Baba et al., 2015b). Another comprehensive expressions study of candidate genes involved carotenoid/ apocarotenoid biogenesis throughout five developmental stages revealed that the expression of MEP/carotenoid transcripts, together with CCD2 and UGT2, but not PDS-II and ZDS, have very high positive correlations (Go´mez-Go´mez et al., 2017). Differential expression of transcription factors such as MYB, MYB-related, WRKY, C2C2-YABBY, and bHLH involved in secondary metabolism indicated that they might have regulatory roles in apocarotenoid biosynthesis and accumulation in C. sativus in a spatio-temporal manner (Baba et al., 2015b; Jain et al., 2016). The expression of CsULT1, an ultrapetala transcription factor, increases gradually in saffron stigma until the anthesis stage, whic is similar to the trend of accumulation of crocin, and therefore, suggesting a regulatory role for the novel transcription factor in apocarotenoid biosynthesis (Ashraf et al., 2015). This anticipation was confirmed by overexpression of CsULT1 in saffron calli (Ashraf et al., 2015). A zinc finger transcription factor, called CsSAP09, was identified in C. sativus; its upstream region contains light and stress responsive elements. CsSAP09 expression was the highest in stigma tissue, the site of apocarotenoid accumulation, at the anthesis stage, suggesting this transcription factor as a potential candidate for regulation of apocarotenoid biosynthesis (Malik and Ashraf, 2017). Using the EST library from mature saffron stigmas generated by D’Agostino et al. (2007) and available mature miRNAs from miRBase (http://www.mirbase.org/), Zinati and coworkers reported two putative miRNAs (miR414 and miR837-5p) that target the genes that coexpressed with genes such as β-LCY and Ɛ-LCY as well as transcription factors and protein kinase in C. sativus. This indicates that these miRNAs may be involved in a regulatory pathway of carotenoid/apocarotenoid biosynthesis in saffron stigma (Zinati et al., 2016).

15.5

Production of saffron metabolites in microorganisms

Synthetic biology strategies have been adopted to produce valuable saffron apocarotenoids in microorganisms. The expression of β-OHase and ZCD1 genes in Chlorella vulgaris, using the Agrobacterium tumefaciens-mediated transformation method, resulted in production of crocetin in the transgenic microalgae (Lou et al., 2016). Some strains of Saccharomyces cerevisiae produce low levels of β-carotene. By introducing the three key enzymes of crocetin biosynthesis, β-OHase, CCD, and ALDH, from different sources of a β-carotene-producing S. cerevisiae strain, the highest amount of crocetin produced in eukaryotic cells was achieved (Chai et al., 2017). Characterization of the key enzymes involved in apocarotenoid metabolism in saffron provides a platform for future biotechnological applications of these genes in other species, especially those with substrates for crocetin formation (Ahrazem et al., 2015). The EVOLVA company was the first to use S. cerevisiae as the host to produce saffron bioactive compounds. There are several patents granted to EVOLVA including WO2011146833, US20140248668, WO2013021261, WO2015162283, WO2015132411, and US20170044552. Due to low amounts of acetyl-CoA enzyme and the low activity of cellular prenyl phosphate in yeast, MEV pathway is heavily regulated and consequently results in very low levels of prenyl phosphate for commercial production of terpenoid molecule in yeast. Patent document WO2011146833 (Hansen, 2016) disclosed the method for producing isoprenoid compounds including zeaxanthin-3-diglucoside, zeaxanthin, and C30 carotenoids in yeast by coexpressing multiple MEV pathway gene analogs that increase prenyl phosphates, or by expressing the nonendogenous enzyme ATP citrate lyase, which leads to high concentrations of acetyl-CoA in cytosol. It claims that the yeast host cell produces at least 25-fold more isoprenoid compound (150 mg g21 dry weight) than unaltered yeast cells. Patent documents US20140248668 (Raghavan et al., 2017) reveal a recombinant, carotenoid-producing host such as S. cerevisiae, expressing CsZCD alone or in combination with recombinant UGTs. It is clear that S. cerevisiae does not have carotenoid pathway, and therefore genes involved in β-carotene synthesis including PDS, GGDP synthase, and β-carotene synthase were also introduced into the yeast. It claims that the host can produce detectable amounts of one or more of the saffron bioactive compounds like crocetin, crocetin dialdehyde, crocin, or picrocrocin. Patent documents WO2015162283 and US20170044552 (Kumar, 2017) disclosed microorganisms producing saffron compounds including hydroxyβ-cylcocitral and picrocrocin. The hosts are engineered to express exogenous genes encoding cytochrome P450, truncated ferredoxin, flavin-dependent ferredoxin reductase, UGT polypeptides. Patent document WO2015132411

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(Kumar, 2017) gives detailed descriptions of the genes arranged in expression cassettes involved in synthesis of saffron compounds including crocetin, crocetin dialdehyde, and crocin or picrocrocin.

15.6

Saffron
microbe interactions

The mutualistic interactions between saffron roots and arbuscular mycorrhizal fungi (AMF) were detected in different regions of saffron cultivation in Khorasan province, Iran. There were mainly Glomus macrocarpus and less frequently Glomus mosseae species. Artificial inoculation of saffron corms with G. macrocarpus resulted in a 26% increase in dry plant weight. Interestingly this inoculation had a similar effect on onion, but not on the bulbs of narcissus and gladiolus, indicating species-dependent interaction (Kianmehr, 1981). Positive effects of Glomus aggregatum, G. mosseae, and Glomus etunicatum were also reported on saffron (Mohebi-Anabat et al., 2015). AMF colonization on saffron increased corm diameter and flower yield (Aimo et al., 2010), and the amounts of carbohydrates, proteins, phenolic compounds, and minerals in the corms (Lone et al., 2016). There are about 40 times more bacteria in the rhizosphere of C. sativus compared with bulk soil (Ambardar and Vakhlu, 2013). Further analysis detected 22 different genera of bacteria in rhizosphere and cormosphere (the soil around the corms); Pseudomonas was the dominant genus among eight different genera in rhizosphere and Pantoea was the dominant genus among six different genera in cormosphere (Ambardar et al., 2014). Bacillus subtilis has been shown to have beneficial effects on saffron production. Inoculation of saffron corms with spores of FZB24 strain of the bacteria sped up corm growth, increased stigma biomass by 12%, and increased picrocrocin, crocetin, and safranal. Interestingly the manner of bacterial inoculation affected the beneficial effects and thus the optimum application method to increase both qualitative and quantitative traits needs further optimization (Sharaf-Eldin et al., 2008). In another experiment, Bacillus amyloliquefaciens was reported to be effective at controlling corm rot caused by Fusarium oxysporum (Gupta and Vakhlu, 2015). A cDNA encoding a novel class of chitinase, Safchi A, was characterized in saffron. This cDNA is mainly expressed in roots and corms, and its expression is induced by elicitor treatment, methyl jasmonate, wounding, and by the fungi F. oxysporum, Beauveria, and Phoma species, suggesting a defense role for the protein. Furthermore, in vitro assays with the recombinant native protein showed chitinolytic and antifungal activity of the Safchia protein (Lo´pez and Go´mez-Go´mez, 2009). CsPR10 encoding an ortholog of Arabidopsis PR10 protein was identified in saffron stigmas. The CsPR10 protein showed inhibitory effects on the growth of F. oxysporum but not on Verticillium dahlia and Penicillium species (Go´mez-Go´mez et al., 2011). Although it is suggested that CsPR10 is involved in defense responses to pathogens, its expression pattern does not support this speculation. Unlike most defense-related genes, CsPR10 is not expressed in leaves and roots, the main organs that are affected by the pathogens; instead, the highest level of its expression was detected in anthers and tepals (Go´mez-Go´mez et al., 2011).

15.7

Molecular response to abiotic stresses

CCDs are enzymes that cleave carotenoids to produce apocarotenoids. Isoforms of this enzyme in C. sativus (CsCCD4) are expressed in response to dehydration stress, and their heterologous expression in Arabidopsis resulted in transgenic plants with longer roots, higher number of lateral roots, more tolerance to salt, oxidative, and dehydration stresses, along with higher expression of ROS-metabolizing enzymes (Baba et al., 2015b). A β-glucosidase from C. sativus, CsBGlu12, which is a vacuole-localized enzyme, results in accumulation of flavonols, and thereby confers resistance to abiotic stresses through ROS scavenging (Baba et al., 2017). An stress-inducible glycosyltransferase (CsGT) was identified in saffron that its ectopic expression in Arabidopsis thaliana enhanced salt and oxidative stress tolerance (Ahrazem et al., 2015). Characterization of CsGT showed that the gene regulates root growth by modulating auxin signaling and cell cycle, thereby, enhancing survival to salt and oxidative stresses (Ahrazem et al., 2015).

15.8

Conclusion

Researchers have started to investigate the developmental biology and biochemistry of saffron, however, these efforts are still not proportional to the high economic importance of this cash crop. Sterility and very low genetic variation, lack of genome sequence, and lack of functional genomics tools are probably the main reasons for slow progress in saffron molecular research. By the fast progresses in the next generation sequencing technologies, it is expected to have

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more genomic and transcriptomic data for saffron available in the near future. Then, developing fast and efficient functional genomics tools including transient and permanent gene silencing and overexpression techniques are necessary. Studies on microbiome assembly in rhizosphere and cormosphere of saffron will help uncover saffron
microbe interactions and also to develop biofertilizers for this valuable crop.

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2400. Jain, M., Srivastava, P.L., Verma, M., Ghangal, R., Garg, R., 2016. De novo transcriptome assembly and comprehensive expression profiling in Crocus sativus to gain insights into apocarotenoid biosynthesis. Sci. Rep. 6, 22456. Kalivas, A., Pasentsis, K., Polidoros, A.N., Tsaftaris, A.S., 2007. Heterotopic expression of B-class floral homeotic genes PISTILLATA/GLOBOSA supports a modified model for crocus (Crocus sativus L.) flower formation. DNA Seq. 18, 120
130. Kianmehr, H., 1981. Vesicular—arbuscular mycorrhizal spore population and infectivity of saffron (Crocus sativus) in Iran. New Phytol. 88, 79
82. Kirby, J., Keasling, J.D., 2008. Metabolic engineering of microorganisms for isoprenoid production. Nat. Prod. Rep. 25, 656
661. Kitazawa, M.S., Fujimoto, K., 2014. A developmental basis for stochasticity in floral organ numbers. Front. Plant Sci. 5, 545. Kumar, A.S., 2017. Methods for Recombinant Production of Saffron Compounds. Google Patents. Lashbrooke, J.G., Young, P.R., Dockrall, S.J., Vasanth, K., Vivier, M.A., 2013. Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family. BMC Plant Biol. 13, 156. Liao, Y.-H., Houghton, P.J., Hoult, J., 1999. Novel and known constituents from Buddleja species and their activity against leukocyte eicosanoid generation. J. Nat. Prod. 62, 1241
1245. Lone, R., Shuab, R., Koul, K., 2016. AMF association and their effect on metabolite mobilization, mineral nutrition and nitrogen assimilating enzymes in saffron (Crocus sativus) plant. J. Plant Nutr. 39, 1852
1862. Lo´pez, R.C., Go´mez-Go´mez, L., 2009. Isolation of a new fungi and wound-induced chitinase class in corms of Crocus sativus. Plant Physiol. Biochem. 47, 426
434. Lou, S., Wang, L., He, L., Wang, Z., Wang, G., Lin, X., 2016. Production of crocetin in transgenic Chlorella vulgaris expressing genes crtRB and ZCD1. J. Appl. Phycol. 28, 1657
1665. Maggi, L., Carmona, M., Del Campo, C.P., Kanakis, C.D., Anastasaki, E., Tarantilis, P.A., et al., 2009. Worldwide market screening of saffron volatile composition. J. Sci. Food Agric. 89, 1950
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633. Mohebi-Anabat, M., Riahi, H., Zanganeh, S., Sadeghnezhad, E., 2015. Effects of arbuscular mycorrhizal inoculation on the growth, photosynthetic pigments and soluble sugar of Crocus sativus (saffron) in autoclaved soil. Int. J. Agron. Agric. Res. 6, 296
304. Moraga, A.R., Nohales, P.F., Pe´rez, J.A.F., Go´mez-Go´mez, L., 2004. Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas. Planta 219, 955
966. Namin, M.H., Ebrahimzadeh, H., Ghareyazie, B., Radjabian, T., Gharavi, S., Tafreshi, N., 2009. In vitro expression of apocarotenoid genes in Crocus sativus L. Afr. J. Biotechnol. 8, 5378
5382. Nisar, N., Li, L., Lu, S., Khin, N.C., Pogson, B.J., 2015. Carotenoid metabolism in plants. Mol. Plant 8, 68
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246.

Section IV

Saffron processing

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Chapter 16

Bioactive ingredients of saffron: extraction, analysis, applications Seid-Mahdi Jafari1, Maria Z. Tsimidou2, Hamid Rajabi1 and Anastasia Kyriakoudi2 1

Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran,

2

Laboratory of Food Chemistry and Technology (LFCT), School of Chemistry, Aristotle University of Thessaloniki (AUTH), Thessaloniki, Greece

Chapter Outline 16.1 Introduction 16.1.1 International classification of saffron stigmas 16.1.2 Iranian trade categories 16.2 Saffron drying methods 16.3 Extraction of saffron bioactive components 16.3.1 Conventional extraction techniques 16.3.2 Novel extraction methods 16.4 Characterization of saffron bioactive compounds 16.4.1 UV-Vis spectrophotometry 16.4.2 High performance liquid chromatography (HPLC)

16.1

261 262 262 264 264 266 270 272 272 273

16.4.3 Gas chromatography-mass spectrometry (GC-MS) 16.4.4 Electronic nose technique 16.5 Applications of saffron bioactive ingredients: from prehistory up to 21st century 16.5.1 Food industry 16.5.2 Pharmaceutical industry 16.5.3 Cosmetics and other sectors 16.6 Conclusion Acknowledgment References

279 279 282 282 283 284 285 285 285

Introduction

There are many factors influencing saffron production yield and quality before the flowering stage including the number of irrigations (3
5 times), amount of rainfall, the minimum and maximum temperature in summer and winter, respectively, applying fertilizer, etc. (Mollafilabi, 2003; Negbi, 2003). Saffron quality is also strongly dependent on postharvest parameters as well as crop management in the field. There are several crucial steps in saffron processing, from picking the flowers in farms to drying of stigmas, that are characterized by local traditional practices (Aghaei et al., 2018; Carmona et al., 2006b; Ordoudi and Tsimidou, 2004). Quality characteristics of saffron are directly affected by the postharvest processing stages. Microbial counts and color strength along with apparent features of the stigmas determine the price of each batch and are significantly process-dependent. The flowering stage of saffron varies from 15 to 25 days and the number of flowers reaches a maximum around 7
10 days after the appearance of the first flower (Mahdavee-Khazaei et al., 2014). The best time for collecting flowers is before sunrise when they are in budding mode, as no sunlight exposure has occurred and the tepals protect stigmas from physical damage during transport to the processing stage (Sepaskhah and Yarami, 2009). With the dawn of the sun, flowers are surrounded with light and temperatures rise, resulting in opening of the buds. This not only reduces the physical strength of the tepals but also the quality of the stigma, especially its color strength. Extended exposure to light and high temperatures causes flowers to be withered and spoiled (Jafari et al., 2016;

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00016-2 © 2020 Elsevier Inc. All rights reserved.

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Khazaei et al., 2016). The stigmas are polluted with pollen from stamens. More importantly stigmas may stick to the stamens, causing the separation process to fail, which downgrades market acceptance.

16.1.1 International classification of saffron stigmas Saffron stigmas are classified based on the physicochemical characteristics including color strength, appearance, style length, and diameter. Table 16.1 summarizes the specifications of the International Standardization Organization (ISO) 3632 for the physicochemical classifications of saffron in filaments, cut filaments, and powder forms. The ISO 3632 is the guide for international transactions and has been adopted by national standardization organizations in the European Union (e.g., AFNOR, EΛOT). As shown in Fig. 16.1A, a stigma consists of a red section containing all three bioactive components of saffron and a white section called the style. The style length, attachment to the stigma, as well as the morphological features of the red section have crucial roles in saffron categorization. For example, in Iran the expression “strong stigma” refers to the ones that are thicker in diameter as well as straight and smooth in length. If a stigma is instead a thin and wavy structure, the stigma is called a “weak stigma” (Fig. 16.1B).

16.1.2 Iranian trade categories Iran, as the biggest producer of saffron, has set national specifications for characterization of saffron (Table 16.2) in addition to the international specifications. Based on Iranian standards, the different types of traded saffron are Sargol, Pooshal, and Daste as detailed below: TABLE 16.1 Physicochemical classification of saffron in filaments, cut filaments, and powder forms. Characteristics

Specification categories

Test method

1

2

3

Moisture and volatile matter (mass fraction), % max, filament, and cut filament

12

12

12

Saffron powder

10

10

10

Total ash (mass), on dry matter, % max

8

8

8

ISO 928 and ISO 3632-2 (2010), Clause 12

Extraneous matter (mass fraction), % max, floral and plant waste

0.5

3

5

ISO 3632-2 (2010), Clause 8

Foreign matter (mass fraction), % max, from nonanimals (other plants)

0.1

0.5

1

ISO 3632-2 (2010), Clause 9

Acid-insoluble ash (mass fraction), %, on dry matter, max

1

1

1

ISO 930 and ISO 3632-2 (2010), Clause 13

Soluble extract in cold water, (mass fraction), on dry matter, % max

65

65

65

ISO 941 and ISO 3632-2 (2010), Clause 11

Flavor strength (expressed as picrocrocin); A1%1 cm 257 nm, on dry matter, minimum

70

55

40

ISO 3632-2 (2010), Clause 14

Min

20

20

20

Max

50

50

50

ISO 3632-2 (2010), Clause 14

200

170

120

ISO 3632-2 (2010), Clause 14

Absent

Absent

Absent

ISO 3632-2 (2010), Clause 16 and/or 17

ISO 3632-2 (2010), Clause 7

Aroma strength (expressed as safranal); A1%1 cm 330 nm, on dry matter

Coloring strength (expressed as crocin); dry matter, minimum Artificial colorants

A1%1 cm

440 nm, on

Source: Based on ISO 3632-1 (2011). Spices, Saffron (Crocus sativus L.). Part 1: Specification, International Organization for Standardization, Geneva.

Bioactive ingredients of saffron: extraction, analysis, applications Chapter | 16

263

FIGURE 16.1 (A) Different classes (grades) of saffron stigma and (B) physical assessment of saffron stigma.

TABLE 16.2 Classification of saffron according to National Standards of Iran. INSO classification

Color strength

Sargol Negin

.220

Pooshal Grade 1 (Negin)

.200

Pooshal Grade 2

.180

Pooshal Grade 3

.150

Pooshal Grade 4 (Daste)

.140

Source: Based on INSO, 259-1 (2013). Saffron, Specification. Iranian National Standards Organization (INSO), Iran.

1. Sargol saffron: Saffron Sargol, also known as “All Red” or “Coupe´” saffron, is the completely red and cut saffron stigma, which does not have any style section attached to its end. The color strength of this saffron type is in the range of 210
260, depending on the production process and the amount of fragmented stigmas. This type of saffron is categorized in three groups as Sargol Negin, Sargol grade 1, and Sargol grade 2, depending on the color strength, stigma length and thickness, and the amount of fragmented stigmas, as shown in Fig. 16.1A.

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a. Sargol Negin saffron: Saffron categorized in this group means that the collected stigma does not have any style section, foreign materials, or fragmentation. Usually, when the saffron comes from the same farm, the color strength of Sargol Negin is slightly less than Pooshal Negin (see below) due to the heat applied during the production process of Sargol Negin compared to Pooshal. The color strength of this saffron type is in the rage of 240
260. b. Sargol grade 1 and 2: The differences between Sargol Negin and Sargol grade 1 and 2 are related to their color strength and stigma appearance. Usually, suppliers differentiate between the saffron grades with the level of fragmented stigmas. It is generally considered that fragile and weak stigmas have inferior coloring strength. The color strength of Sargol grade 1 and grade 2 is in the range of 220
240 and 200
220, respectively. 2. Pooshal saffron: Saffron Pooshal, also known as “Mancha,” is a special class of saffron produced directly from fresh stigma along with Daste type (No. 3 below), while Sargol category is derived from these two types by applying further processes (Fig. 16.1A). The reason for the selection of this name is its bulk nature, which results in a lower bulk density compared to the Sargol type. This physical property plays an important role in packaging design. The stigmas in this saffron type are intermeshed and connected to each other. This physical state is considered as a quality index. The color strength of Pooshal saffron is in the range of 170
280, depending on the size of style attached to the reddish stigma as well as the stigma classification as strong or weak. a. Pooshal Negin saffron: Thick and smooth stigmas without any style section, foreign materials, or fragmented stigmas are categorized in this group. The highest color strength, the best appearance, and the lowest bulk density belong to this category of saffron. As defined by the Iranian National Standard Organization (INSO), the crocins content of this grade is above 200. Generally, this value ranges from 220 to 280 and this may be elevated up to 300, depending on the harvest time, postharvest handling, field management, etc. b. Pooshal grade 1: If the stigmas are thick and smooth in their appearance and a small section of style is attached to them, saffron is called Pooshal grade 1. Based on the ISO and INSO references, the color strength of this grade is approximately 20
30 units less than that of Pooshal Negin, but in practice this difference could be greater. c. Pooshal grade 2: Pooshal 2 is saffron with fragile and weak stigmas and a significant style section. The color strength of Pooshal grade 2 is approximately 20
30 units lower than that of Pooshal grade 1. d. Pooshal grade 3: Pooshal 3 is saffron with stigmas that are more fragile and weak than Pooshal grade 2. The color strength of Pooshal grade 3 is approximately 20
30 units lower than that of Pooshal grade 2. 3. Daste of saffron: This class of saffron known also as “Bunch” consists of the entire filament (stigma and style) and has the lowest color strength. INSO and ISO standards refer to this class as grade 4 and 3, by the coloring strength of 140 and 120, respectively.

16.2

Saffron drying methods

Similar to the other agricultural crops, postharvest processing plays a crucial role in the quality characteristics of saffron and its physicochemical as well as microbiological properties. The drying process is aimed at protecting the crocins by stopping the enzymatic reactions responsible for its biodegradation, which is achieved with temperatures above 60 C for the minimum possible time (Aghaei et al., 2018, 2019). Selecting the most appropriate drying method is one of the important steps in postharvest processing in order to prepare saffron stigmas with the highest amount of crocins, while obtaining the best morphological features. Generally, during the drying process the moisture content of stigmas decreases between 8% and 10%. The methods of saffron drying are traditional (sun drying, dark-air drying, toasting) and modern (such as freeze drying, microwave drying, etc.) and are selected and applied based on available equipment. The main difference in the methods is related to the temperature applied in the process. Table 16.3 summarizes different studies on the drying of saffron and application of various drying techniques (Acar et al., 2011; Maghsoodi et al., 2012; Raina et al., 1996; Tong et al., 2015a).

16.3

Extraction of saffron bioactive components

Scientists in the phytochemical field have tracked a variety of components in saffron stigmas. The researchers revealed that three groups of compounds responsible for color, taste, and aroma are present in dried stigma (Garavand et al., 2019; Sarfarazi et al., 2019). These are crocins, picrocrocin, and safranal, respectively (Fig. 16.2). The red color of stigmas is due to the presence of uncommon hydrophilic carotenoids named crocins, which are glycosyl and gentiobiosyl esters of crocetin, the 8,80 -diapocarotene-8,80 dioic acid, not naturally found free/unesterified

TABLE 16.3 Drying methods of saffron stigma. Drying method

Principles

Advantages/limitations

References

Sun/shade drying

Spreading the fresh saffron stigmas with diameter of 1
10 mm in sun or shade for several hours to 3
5 days.

Prolonged process can destroy the crocins and result in inappropriate morphological features.

Acar et al. (2011), Carmona et al. (2005), Maghsoodi et al. (2012), Raina et al. (1996)

Freeze drying

Spreading the fresh stigmas onto the tray of freeze dryer followed by decreasing the temperature to 240 C until complete freezing. Gradually reducing the chamber pressure in parallel to temperature increase dries the stigma.

High quality of produced saffron in terms of crocins content and morphological feature.

Spreading of a thin layer of fresh stigmas in microwave tray working at different powers.

The shortest operating time and higher retention of crocins in comparison with long drying time processes, like shade drying.

Nonuniform drying

Microwave drying

Acar et al. (2011), Atefi et al. (2004), Kanakis et al. (2004)

Cost intensive and long processing time.

Maghsoodi et al. (2012), Rajabi et al. (2015), Tong et al. (2015a)

High microwave power can negatively affect both bioactive components and morphological features in terms of color and structure. Electric oven

Fresh stigmas are poured into petri dishes followed by running oven at different temperatures.

Short processing time and higher retention of crocins in comparison with long-time drying processes like shade drying.

Carmona et al. (2005), Maghsoodi et al. (2012), Raina et al. (1996), Tong et al. (2015a)

Vacuum oven drying

Same as electric oven except for the difference in the pressure, which is maintained below the atmospheric level.

Short processing time and higher retention of crocins in comparison with electric oven and long drying process like shade drying.

Raina et al. (1996), Tong et al. (2015a)

Toasting

Putting the fresh stigmas into a silk bottom sieve while heat is applied by various sources.

Nonuniform drying.

Kanakis et al. (2004), Raina et al. (1996)

RefractanceWindow (RW) drying

Spreading of a thin layer of fresh stigmas on a membrane/glass on top of boiling water.

RW dryer through Pyrex glass surface and higher temperatures (70 C and 80 C) led to the highest contents of picrocrocin, safranal, and crocins.

Aghaei et al. (2018, 2019)

FIGURE 16.2 Main bioactive components of dried saffron stigmas.

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SECTION | IV Saffron processing

(Esfanjani et al., 2015; Faridi-Esfanjani et al., 2017). As shown in Fig. 16.2 the difference in the type of sugar moieties placed in the positions R1 and R2 results in the six classes of crocins including crocin 1, crocin 2, crocin 20 , crocin 3, crocin 4, and crocin 5. In this regard, glycosyl and gentiobiosyl moieties along with
H are indwelled in R1 and R2 at different patterns, making various types of crocins (Sarfarazi et al., 2015). Regarding isomeric structure of crocins occurring in saffron, both isomeric structures of cis and trans are present in saffron. Total crocins are measured by reading absorbance of saffron extract with a standard concentration using a UV-vis spectrophotometer at wavelength of 440 nm (Mehrnia et al., 2016, 2017). The bitter taste of saffron comes from a monoterpene glucoside named picrocrocin (C16H26O7, 4-(b-Dglucopyranosyloxy) 2 2,6,6-trimethyl-1-cyclohexene-l-carboxaldehyde). This component not only brings a charming bitter taste but also acts as a precursor for safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde), an essential oil responsible for the saffron aroma (Shahi et al., 2016). Safranal makes up 70% of the total volatile constituents of saffron. It has been reported that this carboxaldehyde volatile component is present in saffron due to the heat applied during the drying process and also because of enzymatic activity of ß-glucosidase on picrocrocin. Researchers have proposed another pathway in which picrocrocin, due to loss of glucose molecule, is converted into oxysafranal (HTCC, 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene), an intermediate volatile precursor of safranal. HTCC is also converted into safranal through release of a molecule of water. Several techniques have been developed in order to extract saffron bioactive components with the highest quality while obtaining the highest extraction efficiency (Garavand et al., 2019). Up to now, a variety of methods including high hydrostatic pressure extraction (Shinwari and Rao, 2018), solid-phase microextraction (SPME) (D’Archivio et al., 2018), supercritical fluid extraction (SFE) (Nerome et al., 2016), maceration (Rajabi et al., 2015; Sarfarazi et al., 2015), microwave-assisted extraction (Jafari et al., 2019; Nescatelli et al., 2017), subcritical water extraction (SWE) (Sarfarazi et al., 2019),ultrasonic solvent extraction (Maggi et al., 2011), thermal desorption (TD) (Alonso et al., 1996; Kyriakoudi and Tsimidou, 2015), hydrodistillation (HD) (Kanakis et al., 2004; Maggi et al., 2011), microsimultaneous hydrodistillation
extraction (MSDE) (Ro¨del and Petrzika, 1991; Tarantilis and Polissiou, 1997), and vacuum headspace (Tarantilis and Polissiou, 1997) have been carried out in order to obtain whole saffron extract or with the aim of purification and separation of a specified saffron bioactive component. Due to the presence of compounds of both hydrophilic and lipophilic nature in saffron, selecting the appropriate solvent as well as other process variables such as extraction time and temperature are crucial determinants of the saffron extract quality and quantity. In this regard, the hydrophilic nature of crocins and picrocrocin results in the selection of water and alcohol as the solvents of choice, while safranal can be extracted well with less polar solvents such as diethyl ether and petroleum ether. Many researchers have investigated the role of extraction technique, solvent type, temperature, saffron stigma physical state, extraction time, ratio of saffron to solvent, etc., on the extraction efficiency (Jalali-Heravi et al., 2009; Kyriakoudi et al., 2012; Kyriakoudi and Tsimidou, 2015, 2018a; Mohajeri et al., 2010; Nerome et al., 2016; Nescatelli et al., 2017; Rubert et al., 2016; Sa´nchez et al., 2009; Sarfarazi et al., 2015; Sereshti et al., 2014; Shinwari and Rao, 2018; Tong et al., 2018). When the isolation of crocins is desired, the main limitation in designing an extraction system is the sensitivity of crocins to degradation in aqueous media. In this section, some of the extraction methods are reviewed briefly and a summary of extraction studies on saffron bioactive ingredients has been presented in Table 16.4.

16.3.1 Conventional extraction techniques This class of extraction methods is usually based on the power of different solvents in use and the application of heat and/or mixing. The methods of maceration, Soxhlet, and HD are placed in this category.

16.3.1.1 Maceration The most common method for extraction of whole saffron extract is maceration. The solvent extraction method is initiated by mixing an appropriate amount of saffron and solvent, followed by stirring over a predetermined time and speed, and ended by filtration and reading the absorbance of the resulting extract using a UV-Vis spectrophotometer. The ISO method for quality control of saffron is based on the maceration method (Sarfarazi et al., 2015). In ISO instructions, assessment of the quality of saffron is conducted by reading the absorbance of saffron extract at 257, 330, and 440 nm after preparation as follows: 500 mg of powdered saffron is transferred into a volumetric flask (1000 mL) containing 900 mL distilled water, stirred (1000 rpm, 1 hour) and the volume brought to 1000 mL. Next, 20 mL of this extract is transferred into a 200 mL volumetric flask and diluted with water to the mark. The results of work by Orfanou and Tsimidou (1995) on the relevant parameters in saffron extract coloring strength prepared via the maceration method

Bioactive ingredients of saffron: extraction, analysis, applications Chapter | 16

267

TABLE 16.4 An overview of different studies dealing with extraction of saffron bioactive compounds. Extraction method

Parameters

Solid liquid extraction

G

G

Microwaveassisted extraction

G

G G

G

Analyte of interest

Results

Solvent type (water, methanol, acetonitrile, ethanol, methanol/ water (50/50, v/v), acetonitrile/ water (75/25, v/v) and ethanol/ water (70/30, v/v)) Ultrasound effect on EF

All bioactive components of saffron

G

Solvent type (methanol, acetone, diethyl ether, dichloromethane, ethanol, ethylacetate, methanol/ water (50/50, v/v), ethanol/water (50/50, v/v)) Extraction temperature Extraction time (1, 10, and 19 min) Extraction volume (2 and 10 mL of solvent)

All bioactive components of saffron

G

G

G

G

Ultrasoundassisted extraction

G

G

G

Ultrasoundassisted extraction

G

Ultrasoundassisted extraction

G

G

G

G

References

The best solvent was methanol/ water (50/50, v/v) followed by ethanol/water (70/30). Ultrasound increased the extraction efficiency.

Rubert et al. (2016)

The best solvent, time, temperature, and volume for obtaining the highest concentration of safranal was ethanol/water (50/50, v/v), 1 min, 40 C and 10 mL, respectively. The best solvent, time, temperature, and volume for obtaining the highest concentration of crocin 1 were methanol/water (50/50, v/v), 10 min, 40 C, and 10 mL, respectively. The best solvent, time, temperature, and volume for obtaining the highest concentration of crocin 1 were methanol/water (50/50, v/v), 1 min, 40 C, and 10 mL, respectively.

Jafari et al. (2019), Nescatelli et al. (2017)

Solvent type (hexane and chloroform) Time of extraction (15, 30, and 60 min) The concentration of saffron in each organic solvent (20, 40, and 60 g L21)

Safranal

G

The best solvent, time, and concentration of saffron in each organic solvent for obtaining the highest concentration of safranal were chloroform, 15 min, and 20 g L21 of saffron, respectively.

Maggi et al. (2011)

Saffron: solvent ratio [182, 425, 1013, 1600, and 1843 (w/v)] Duration of sonication (5, 9, 18, 26, 30 min)

Total crocetinesters

G

The best saffron: solvent ratio and sonication time for obtaining the highest concentration of crocetin were 1600 and 9 min, respectively.

Kyriakoudi and Tsimidou (2015)

Percentage of methanol [0.4, 10.5, 25.2, 40, and 50 (%, v/v)] Duration of sonication (1.2, 7, 15.5, 24, 29.8 min) Duty cycles of sonication (active interval) (0.2, 0.3, 0.5, 0.7, and 0.8 s)

Crocins and picrocrocin

G

The best methanol concentration, sonication time and duty cycles for obtaining the highest concentration of crocins were 50%, 30 min, and 0.2 s on/0.8 s off, respectively. The best methanol concentration, sonication time, and duty cycles for obtaining the highest concentration of crocins and picrocrocin were 44%, 30 min, and 0.6 s on/0.4 s off, respectively.

Kyriakoudi et al. (2012)

G

(Continued )

268

SECTION | IV Saffron processing

TABLE 16.4 (Continued) Extraction method

Parameters

Maceration

G G

Analyte of interest

Results

Solvent type (hexane, methanol) Different drying methods [oven (43 C for 100 min, 80 C for 20 min then 43 C for 70 min, 87 C for 20 min then 43 C for 70 min, 75 C for 20 min then 43 C for 70 min, 43 C for 100 min, 92 C for 20 min then 43 C for 70 min), food dryer (46 C for 60 min, 58 C for 20 min then 46 C for 40 min); fresh/no drying; frozen/no drying; frozen, thawed, and then dried (87 C for 20 min then 43 C for 70 min)]

All bioactive components of saffron

G

G

G

References

The best solvent and drying method for obtaining the highest concentration of safranal were hexane and oven drying (87 C for 20 min then 43 C for 70 min), respectively. The best solvent and drying method for obtaining the highest concentration of HCCa were hexane and frozen/no drying, respectively. The best solvent and drying method for obtaining the highest extraction efficiency (%) of picrocrocin were methanol and fresh/no drying, respectively.

Gregory et al. (2005)

Maceration

G

Solvent type (ethyl ether, acetone, acetonitrile, methanol, ethanol, isopropanol, ethanol-water (50% v/v), and water)

Picrocrocin, HTCC, and crocins

G

The best solvent for picrocrocin, HTCC, and crocins extraction was water followed by ethanol:water (50:50).

Iborra et al. (1992)

Emulsion liquid membrane

G

Surfactant type (Span 80, ENJ3029, plyamine type-surfactants) Membrane type (CCl4, N-decane, CH3Cl, toluene) Surfactant concentration (0.5, 2.5, 7, 7.5, 10) Treat ratio (0.1, 0.2, 0.3, 0.4) Phase ratio (0.4, 0.6, 0.8, 1.0, 1.2) Stirring rate (100, 200, 300, 400, 500)

All bioactive components of saffron

G

The best surfactant, membrane type, surfactant concentration, treat ratio, phase ratio, and stirring rate for obtaining highest extraction efficiency were Span 80, N-decane, 2.5%, 0.3, 0.8, and 300 rpm, respectively.

Mokhtari and Pourabdollah (2013)

Solvent type (methanol: ethylacetate, methanol: ether, methanol: chloroform) Solvent volume (10, 20, 30, 40, 50 mL) Extraction time (15, 37.5, 60, 82.5 min) Extraction step (1 and 4) Solvent ratio (10, 30, 50, 70, 90) Sample amount (0.5, 1.0, 1.5, 2.0, 2.5 g)

Volatile components of saffron

G

The best solvent was methanol: ethylacetate and the optimum values of factors were: 2.38 g sample, 29.04 mL solvent, 69.23% methanol solvent ratio, and 71.8 min for the extraction time. The effect of extraction steps on the response was not significant.

Jalali-Heravi et al. (2009)

Voltage Pulse width Pulse numbers

All bioactive components of saffron

G

The optimum condition was voltage of 5 kV, pulse number of 100 pulses, and pulse width of 35 ms.

Pourzaki et al. (2013)

Ethanol concentration (0%
100%) Extraction time (2
7 h) Temperature (5 C
85 C)

All bioactive components of saffron

G

The optimum conditions in terms of obtaining highest amount of saffron bioactive component was ethanol concentration of 33.33%, extraction time of 2.0 h, and temperature of 85.0 C.

Khazaei et al. (2016), Sarfarazi et al. (2015)

G

G

G G G

Ultrasonicassisted solvent extraction

G

G

G

G G G

Pulsed electric field extraction

G

Maceration

G

G G

G G

G

(Continued )

Bioactive ingredients of saffron: extraction, analysis, applications Chapter | 16

269

TABLE 16.4 (Continued) Extraction method

Parameters

Different extraction methods

G

Supercritical CO2 extraction

G

G G

Analyte of interest

Results

Extraction method [solventassisted flavor evaporation (SAFE), liquid
liquid extraction, solidphase extraction, and simultaneous distillation extraction]

Safranal

G

According to sensory analysis, the aromatic extract obtained by SAFE was the most representative of saffron odor.

Amanpour et al. (2015)

Temperature (40 C, 60 C, and 80 C) Pressures (20, 30, 40 MPa) Enrainer type (water and methanol)

All bioactive components of saffron

G

The best entrainer for extraction of picrocrocin, safranal, and HTCC was ethanol. Water was the best type for extraction of deglycosylated crocin and α-crocin. The optimum pressure for extraction of α-crocin, picrocrocin, and deglycosylated crocin was 30 MPa and for the rest was 40 MPa. The optimum temperature for extraction of all components was 80 C.

Nerome et al. (2016)

The optimal values of variables for safranal extraction were 92 C, 21.3 MPa, 0.9 cm3 min21 and 122.0 min. The optimal values of variables for crocin were obtained at 44 C, 19.3 MPa, 1.0 cm3 min21, and 110.0 min.

Goleroudbary and Ghoreishi (2016)

The best system based on their high top phase recovery yield and low cost of system constituents was ethanol
potassium phosphate ATPS. The best conditions of ATPS were volume ratio 5 3.2, ethanol 19.8% (w/w), potassium phosphate 16.5% (w/w), TLL of 25% (w/w), 0.1M NaCl, and 2% (w/w) of sample load.

MontalvoHerna´ndez et al. (2012)

G

G

Supercritical CO2 extraction

G G G

G

Aqueous two-phase system (ATPS)

G

G

G G

G

G

G

G

G

Temperature (35 C
105 C) Pressure (10
30 MPa) SC-CO2 flow rate (0.3
1.5 cm3 min21) Dynamic extraction time (30
150 min)

Safranal and crocins

Different types of ATPS (polymerpolymer, polymer-salt, alcoholsalt, and ionic liquid-salt) Polyethylene glycol (PEG) molecular mass (400, 1000, 3350, 8000, and 10,000 g mol21) Volume ratio (0.33
7.25) PEG concentration (3.5%
20.2% w/w) Salt (potassium phosphate) concentration (10.4%
20% w/w) Dextran concentration (8.2%
15.5% w/w) Ionic liquid concentration (13%
35.2% w/w) Ethanol concentration (14%
17% w/w) TLL (15%
50% w/w)

Crocins

G

G

G

G

References

Macroporous resins

G

Resin type (AB-8, Amberlite XAD1180, XAD-1600, EXA-117, EXA32, EXA-45, EXA-50, EXA-118, HP20 and HPD-100A)

Crocins of gardenia fruit

G

The best resins in terms of adsorptive capacity and selectivity for crocin were XAD1180, HP20, HPD-100A, and AB8 (best).

Yang et al. (2009)

Macroporous resins

G

Resin type [Polystyrene (D101, X5, LX60, AB-8, LX38, LX28, LX8) and Acrylate (LX17)]

Crocins of gardenia fruit

G

The best resin based on static absorption/desorption experiments was LX60.

Feng et al. (2014) (Continued )

270

SECTION | IV Saffron processing

TABLE 16.4 (Continued) Extraction method

Parameters

Solid-phase extraction

G

G

G

a

Polymer type [gentiobiose imprinted polymer (Gent-MIP) and blank nonimprinted polymer] Solvent used for binding study [water, acetic acid 2% (v/v), acetic acid 5% (v/v), acetic acid 10% (v/v)] Type of washing solvents (methanol, THF, water, and ACN)

Analyte of interest

Results

Crocins of saffron

G

G

G

The inclusion of gentiobiose in polymer increased the affinity of the Gent-MIP for the crocin than the nonimprinted one. The best solvent for binding was acetic acid 2% (v/v). The best washing solvent was ACN.

References Mohajeri et al. (2010)

2 4R-hydroxy-ß-cyclocitral.

showed that the process duration, type of solvent and filter, as well as the stage in which the extract was filtrated played a significant role. In this regard, by increasing the pore size of filter paper or processing time (up to 24 hours), as well as by filtering the extract prior to final dilution, the coloring strength was decreased significantly. Also, it is reported that the best solvent in order to extract the highest amount of saffron active components is methanol (50% v/v) followed by ethanol (50% v/v) and water.

16.3.1.2 Soxhlet extraction In this method, the dried saffron is placed in an extraction thimble and extracted using an appropriate solvent. Successive and exhaustive Soxhlet extraction is utilized for extraction of picrocrocin using light petroleum, diethyl ether, and methanol as solvents (Tarantilis et al., 1994). In this work, nonglucoside carotenoids and lipids, lipids and picrocrocin, and the glucoside carotenoids were found in light petroleum extract, diethyl ether extract, and methanol extract, respectively. Methanol is the preferred solvent for extraction of saffron components and the resulting extract has been used for medicinal, pharmaceutical, and food purposes (Goleroudbary and Ghoreishi, 2016). On the other hand, Feizzadeh et al. (2008) and Samarghandian et al. (2013) worked on preparation of aqueous saffron extract using Soxhlet by adding 15 g powdered saffron and 100 mL distilled water into the extractor for 18 hours.

16.3.1.3 Hydrodistillation As its name implies, HD is a process of volatile component isolation in which water vapor penetrates herbal cells and acts as a carrier of volatiles to a condenser rod. In this method, the dried saffron stigma is soaked in water (or a mixture of water and alcohol) for a period of time followed by heating the mixture to the boiling point. Volatiles are carried away in the steam to a condenser that is cooled by a stream of water, liquefying the compounds for collection. Kanakis et al. (2004) worked on quantification of safranal using MSDE. They used diethyl ether as a solvent and a mixture of water/glycol (210 C) to cool the condenser. The MSDE procedure was carried out for 2 hours.

16.3.2 Novel extraction methods 16.3.2.1 Supercritical fluid extraction The cornerstone of this method is based on passing a supercritical fluid through the sample. Carbon dioxide is the most common supercritical fluid due to its characteristics of interest including nontoxicity and nonflammability, liquid-like density, low viscosity, high diffusivity, and selective extraction ability (McHugh and Krukonis, 2013). The SFE based on carbon dioxide (SFE-CO2) works well with less polar components (safranal in the case of saffron). In order to increase the scope of the process toward polar components extraction, water or an organic solvent is introduced as an entrainer. When water is used in the SC-CO2 system, two factors are taken into account: (1) the solubility of the components of interest is increased due to the impact of water as a entrainer and (2) water sorption of plant tissue followed by swelling resulted in components of interest escaping more rapidly and easily (Nerome et al., 2016). Lozano et al. (2000) conducted a procedure based on SFE-CO2 to isolate safranal from saffron and reported extraction of safranal and a small amount of 4-hydroxy-2,6,6-trimethyl-1-carboxaldehyde-1-cyclohexane. Nerome et al. (2016) used a SFE-

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271

CO2 system with water and methanol as entrainers to extract saffron bioactive compounds. Their results revealed that the efficiency of the SFE-CO2 system in extraction of both hydrophilic and lipophilic components of saffron was significantly higher than the maceration method via water and methanol. Also, using methanol as an entrainer maximized extraction yield of picrocrocin and safranal, while maximum extraction of crocins was achieved through running the system with water as an entrainer.

16.3.2.2 High pressure extraction The principles of high pressure extraction (HPE) and high pressure processing of food are similar and based on applying a pressure between 100 and 1000 MPa for a determined time. Like other modern methods, HPE brings advantages of lower energy consumption, and the produced extract meets the highest quality indexes. Unlike thermal extraction methods, in the HPE process the covalent bonds remain intact and hydrophobic, hydrogen, and ionic bonds are affected (Li et al., 2012; Shinwari and Rao, 2018; Shouqin et al., 2004). Shinwari and Rao (2018) assessed extraction of nutraceuticals from saffron using the thermal-assisted high hydrostatic pressure process by applying pressure of 100
600 MPa in combination with elevated temperatures (30 C
70 C). In this work, saffron powder and water were mixed in a ratio of 1:100w/v and vacuum-packed followed by hydration and exposure to high pressure. They revealed that the extraction efficiencies of crocin, picrocrocin, and safranal were increased significantly (52%
63%, 54%
85%, and 55%
62%, respectively) when compared to the maceration method. The pressure is transmitted through a solution of monopropylene glycol (30%). Buzrul et al. (2008) used water, ethylene glycol, and ethanol as pressure transmitting fluids to treat liquid foods by HPP.

16.3.2.3 Ultrafiltration Ultrafiltration (UF) is a separation process in which membranes with pore size of 0.1
0.001 μm are used to remove high-molecular-weight substances, colloidal materials, and organic and inorganic polymeric molecules. The attractive properties of UF include its mild operating conditions and relatively high selectivity, making it a popular method toward conventional one. UF efficiency is affected by a variety of factors including total soluble solid content, nominal molecular weight limit, fouling behavior, and cross-flow rate (Zeman and Zydney, 2017). Sa´nchez et al. (2009) investigated the effect of a centrifugal UF process on saffron bioactive components. In that work, first, the hydrophobic components of saffron were removed, then aqueous saffron extract was prepared. The extract was then centrifuged and subjected to UF. Successive dead-end microfiltration and cellulose acetate membrane filters were finally used to clarify the end product.

16.3.2.4 Microextraction methods One of the most important features of the microextraction method is use of negligible amounts of solvent, resulting in their reputaiton as “green technology.” Other attractive characteristics of this category are simplicity, high efficiency, and short processing time (Ocan˜a-Gonza´lez et al., 2016). The methods of SPME, stir-bar sorptive extraction (SBSE), and dispersive liquid
liquid microextraction (DLLME) are discussed in the following subsection.

16.3.2.5 Solid-phase microextraction SPME is a comparatively new extraction method and is a well-known solvent-free technique with characteristics such as high speed, ease of use, and sensitivity. SPME is the process in which the components of interest (in gaseous or liquid phase) are absorbed on a fiber coating. Identification of these components can be achieved successively through gas chromatography (Pawliszyn, 2011). D’Archivio et al. (2018) analyzed the aroma profile of saffron using SPME as follows: saffron hydration was performed, followed by conditioning of fiber and exposure to SPME. Gas chromatography (GC) was used to identify the analyte at 250 C for 5 minutes.

16.3.2.6 Stir-bar sorptive extraction Like SPME, SBSE requires only a small amount of solvent to extract biochemicals from the sample. In this method, the desired component is moved into a nonmiscible liquid phase. Polydimethylsiloxane (PDMS) is the predominant sorptive extraction phase due to its thermo-stable properties and possession of desired diffusion manner (Prieto et al., 2010). Maggi et al. (2011) used SBSE to assess semivolatile organic contaminants and pollutants in saffron as follows. PDMS coated stir bars were stirred (1000 rpm, 25 C, 14 hours) through the saffron aqueous solution containing methanol,

272

SECTION | IV Saffron processing

sodium sulphate anhydrous, and methanolic solutions. After that, the stir bar was separated followed by rinsing and drying. The analytes were assessed by introducing them into a TD tube for GC/MS/MS analysis.

16.3.2.7 Dispersive liquid
liquid microextraction DLLME as a novel extraction method is at the center of attention due to its positive characteristics such as simplicity, low cost, and ease of running and development. DLLME is conducted through formation of a ternary solvent system consisting of dispersing solvent, extracting solvent, and the aqueous medium that contains the analyte. Each part of this system plays a specified role in order to extract the maximum amount of analyte. Accordingly, the extraction solvent is scattered through the aqueous medium by acting as a dispersing solvent. DLLME is also applied as a sample preparation technique because of its ability to give high enrichment factors by treating a relatively negligible amount of aqueous sample. This process includes the following steps (Rezaee et al., 2006, 2010): 1. The binary system of extracting and dispersing solvents is introduced to an aqueous medium. 2. Dispersion of analyte and extracting solvent is achieved in a short time, named cloudy state. 3. Centrifugation is applied to isolate dispersion solvent enriched with analyte followed by analysis with a microsyringe. Sereshti et al. (2014) aimed to isolate and enrich saffron volatile components through ultrasound-assisted extraction (UAE) in conjunction with DLLME. In this work, a mixture of methanol and acetonitrile was added into a specified amount of ground saffron and exposed to an ultrasonic process. Henceforth, preconcentration solvent (chloroform) is introduced into the centrifuged extract followed by quick injection into aqueous NaCl solution. Accordingly, cloudy state appeared and the dispersion solvent was separated using centrifugation. Finally, GC was used to analyze the extracted analyte.

16.4

Characterization of saffron bioactive compounds

16.4.1 UV-Vis spectrophotometry Detection and estimation of the content of crocins, picrocrocin, and safranal in a saffron sample has been carried out until now almost exclusively with the aid of UV-Vis spectrophotometry. This technique is included in the ISO 3632-2 (2010), which is related to the determination of the major saffron constituents (Rajabi et al., 2019). In particular, according to this standard, after the preparation of an aqueous saffron extract, a spectrum is recorded in the range of 200
700 nm. Absorbance values at specific wavelengths of 440, 250, and 330 nm are used to estimate of the content of crocins, picrocrocin, and safranal in a saffron sample as measures of “coloring,” “flavor,” and “aroma” strength, respectively, using Eq. (16.1). E1% λmax 5

D 3 10; 000 mð100 2 HÞ

(16.1)

where D is the absorbance value at 440 nm (coloring strength), 330 nm (aroma strength), and 250 nm (flavor strength); m, the mass of the test portion (g); and H, the moisture and volatile content of the sample (%, w/w). Orfanou and Tsimidou (1996) proposed the exploitation of the entire UV-Vis spectrum instead of only specific wavelengths using derivative spectroscopy. Especially a second derivative spectrum can be a valuable tool for the accurate determination of λmax and for the resolution of overlapping peaks. Representative zero order and second derivative spectra of authentic Greek, Spanish, and Iranian authentic saffron samples are shown in Fig. 16.3. Derivative UV-Vis spectrophotometry has been used by Zalacain et al. (2005) to detect the presence of various artificial colorants, namely naphthol yellow, tartrazine, quinoline yellow, Sunset yellow, Allura red, amaranth, azorubine, Ponceau 4R, and Red 2G, in saffron. Masoum et al. (2015) exploited the second derivative to simultaneously detect the presence of two colorants with a high degree of overlapping UV-Vis spectra, tartrazine and sunset yellow, in adulterated saffron. In another work, Ordoudi et al. (2017) found that the second derivative spectrum of authentic saffron changed substantially with the addition of 2% w/w of carminic acid, a natural colorant that can be illegally added to saffron to enhance coloring strength. The authors suggested that the second derivative could provide a clue about the presence of carminic acid in saffron even at relatively low amounts. Doubts about whether or not absorbances at 257 and 330 nm are representative of picrocrocin and safranal content, respectively, have been expressed by various researchers working in the field of saffron since the 90s (e.g., Orfanou and Tsimidou, 1996). In particular, absorbance at 257 nm has been frequently criticized by numerous researchers as

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273

FIGURE 16.3 Representative zero order and second derivative spectra of Greek, Spanish, and Iranian authentic saffron samples. Data from the saffron sample collection of LFCT.

being related to glycosidic bonds of crocins rather than to picrocrocin and absorbance at 330 nm as being related to ciscrocins rather than to safranal. Garcia-Rodriguez et al. (2017) prepared aqueous extracts of 390 saffron samples and analyzed them both by UV-Vis spectrometry and high performance liquid chromatography
diode array detector (HPLC-DAD). Quantification of safranal in both cases was carried out with proper calibration curves at 330 nm. The authors observed no correlation between the safranal content found with UV-Vis and HPLC-DAD analysis and suggested that this was due to an overestimation in safranal content using the UV-Vis that was related to the simultaneous absorbance of cis-crocins at 330 nm. Moreover, considering the differences in polarity among crocins and picrocrocin as well as safranal, the first two are indeed expected to be present in an aqueous saffron extract prepared according to ISO 3632-2. In contrast, nonpolar safranal is not expected to be fully extracted with water (Kyriakoudi and Tsimidou, 2018b). Nevertheless, currently many researchers continue to use the extraction protocol proposed by ISO 3632-2 followed by UV-Vis analysis for the determination of crocins, picrocrocin, and safranal. To overcome the limitations regarding the extraction of safranal with water, different nonpolar solvents in combination with various extraction means have been reported in the literature. In particular, Maggi et al. (2011) prepared saffron extracts by means of UAE using diethyl ether or chloroform as extraction solvents. The authors compared the content of safranal obtained by measuring the absorbance of the prepared extracts at 330 nm using UV-Vis spectrometry to its content obtained by GC-MS and found a good correlation. It is worth mentioning that besides the abovementioned limitations, UV-Vis spectrophotometry finds many applications still today for various purposes. For instance, Cossignani et al. (2014) examined the effect of drying conditions of saffron produced in the Umbria region (Italy) on the content of crocins, picrocrocin, and safranal using the E1% values obtained by UV-Vis spectrophotometry. Moreover, D’Archivio and Maggi (2017) applied principal component analysis (PCA) to the UV-Vis spectra of aqueous extracts of 81 saffron samples from various areas in Italy for their geographical classification.

16.4.2 High performance liquid chromatography (HPLC) In the context of the revision of the ISO 3632 standard in 2003 (ISO 3632-2, 2003), HPLC coupled to a UV-Vis detector was introduced for the first time. However, it is worth mentioning that its use was restricted only to the detection of artificial acidic colorants in saffron and was not proposed for apocarotenoid analysis. According to the standard,

274

SECTION | IV Saffron processing

TABLE 16.5 Gradient elution system for saffron analysis. Stage

Time (min)

Solvent A

Solvent B

Solvent C

Equilibration

10

90

10

0

1

0

90

10

0

2

7

48

52

0

3

10

48

52

0

4

14

0

60

40

5

24

0

60

40

6

25

90

10

0

Source: Based on ISO 3632-2 (2010). Saffron (Crocus sativus L.). Part 2: Test Methods. Organization for Standardization, Geneva.

chromatographic separation was carried out on a C18 column (25 cm 3 4.0 mm, 5 μm). As mobile phase, either an aqueous solution of 1 mM tetra-n-butylammonium hydrogen sulfate and 1 mM potassium dihydrogen phosphate (pH 5 4.5) (A) and acetonitrile (B) (70:30, v/v) or an aqueous solution of 1.4 mM tetra-n-butylammonium hydrogen sulfate and 1.4 mM potassium dihydrogen phosphate (pH 5 4.5) (A) and acetonitrile (B) were proposed. The flow rate was 1 mL min21. Separation of saffron compounds could be performed with either isocratic or gradient elution [0
14 minutes, 0% (B)], 14
30 minutes, 100% (B), 30
40 minutes, 100% (B). In the latest revision of the standard in 2010 (ISO 3632-2, 2010), the use of a C18 chromatographic column (150 mm 3 4.6 m, 3 μm) was proposed along with a guard column (10 mm 3 4.6 mm, 4 μm). As mobile phase, an aqueous solution of 0.01 M potassium dihydrogen phosphate (pH 5 7) (A), methanol (B), and acetonitrile (C) were proposed. Separation was carried out at a flow rate of 0.8 mL min21 using gradient elution as described below (Table 16.5). It is quite strange that although HPLC has been the main analytical tool for the analysis of saffron apocarotenoids in literature, it has not yet been introduced in the standard for this purpose. Alterations regarding the chromatographic column characteristics as well as the elution gradient system are numerous. Table 16.6 gives an overview of the chromatographic systems and conditions that are reported in literature for the analysis of crocins, picrocrocin, and safranal up to today. More specifically, separation of saffron apocarotenoids is carried out on RP C18 chromatographic columns with length that ranges from 100 to 250 mm, internal diameter from 2.1 to 4.9 mm, and particle size in the range of 4
10 μm. Analysis is usually carried out at ambient temperature, even though higher temperatures of 30 C have been also reported. Gradient elution is almost exclusively used for the separation of saffron apocarotenoids. The mobile phase generally includes water and polar organic solvents such as methanol or acetonitrile. The addition of acid such as acetic, phosphoric, or formic acids (0.25%
1%) are suggested to regulate the pH in order to improve peak resolution by inhibiting the ionization of compounds during analysis. UV-Vis and DADs, coupled or not to a mass detector (MS), are usually used for the detection of crocins, picrocrocin, and safranal. The characteristic wavelengths for the detection of these compounds are usually 440, 250, and 330 nm, respectively. A typical RP-HPLC-DAD profile is shown in Fig. 16.4. Spectral characteristics, λmax values, as well as the major ions of the individual apocarotenoids are presented in Table 16.7. It is worth mentioning that the exploitation of UHPLC has been reported in literature for the rapid separation and identification of saffron compounds. In particular, Rocchi et al. (2018) used a UHPLC-MS/MS system equipped with a reversed-phase Kinetex C18 column (100 mm 3 2.1 i.d., 1.7 μm) packed with Core Shell particles in order to separate the trans- and cis-isomers of crocins of 42 saffron samples with different origin, age, and drying conditions. The mobile phase consisted of water (5 mM formic acid) (A) and acetonitrile (5 mM formic acid) (B) with a flow rate of 0.3 mL min21. The authors managed to separate crocins within 10 min. In the same content, Moras et al. (2018) proposed a UHPLC-DAD/MS method for the quality assessment of saffron as well as for the detection of adulteration with Gardenia jasminoides Ellis. A Core-Shell reversed-phase Kinetex C18 column (150 mm 3 2.1 i.d., 2.6 μm) was used for the analysis. The mobile phase consisted of water (0.01% formic acid) (A) and acetonitrile (B) with a flow rate of 0.6 mL min21. 35 compounds including trans- and cis-crocins, picrocrocin, as well as kaempherol derivatives were identified. The authors suggested that with this method it was possible to separate and identify compounds that are not

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275

TABLE 16.6 Overview of the chromatographic systems and analytical conditions for crocetin sugar esters, picrocrocin, and safranal determination. Aim of the study

Chromatographic column

Mobile phase

Elution conditions

Detector

Reference

Development of an HPLC/PDA/ESIMS method for the identification of safflower, marigold, and turmeric in saffron

Gemini C18

Gradient elution

0 min, 95%
5% B

Sabatino et al. (2011)

150 mm 3 2.1 i.d, 3 μm

A: H2O (0.3%, v/v, formic acid), B: ACN (0.3%, v/v, formic acid),

0
50 min, 72%
28% B

DAD (520, 440, 425, 410, 350, 250 nm)

50
60 min, 57%
43% B

Flow rate 0.2 mL min21

60
65 min, 57%
43% B

MS (ESI, negative ion mode)

65
80 min, 95%
5% B Development of a UAE method for the recovery of crocetin sugar esters and picrocrocin from saffron

LiChroCART Superspher 100 C18 125 3 4 mm i. d., 4 μm

Linear gradient elution

20 min, 20%
100% B

DAD (440, 330, 250 nm)

Kyriakoudi et al. (2012)

DAD (440, 250 nm)

Cossignani et al. (2014)

A: H2O (0.1%, acetic acid), B: ACN Flow rate 0.5 mL min21

Characterization of secondary metabolites in saffron from central Italy

Development of a HPLC-DAD method for the determination of the major saffron apocarotenoids from an aqueous extract prepared according to ISO 3632

Analysis of bioactive saffron constituents

Gemini C18

Gradient elution

0
5 min, 90%
10% B

100 mm 3 2.0 i.d, 5 μm

A: H2O, B: ACN

5.25 min, 20%
80% B

Flow rate 0.8 mL min21

25
30 min, 20%
80% B

MS (ESI, negative and positive ion modes)

Phenomenex Luna C18

Gradient elution

0
5 min, 80%
20% B

150 mm 3 4.6 i.d., 5 μm

A: H2O, B: ACN

5
15 min, 20%
80% B

DAD (440, 330, 250 nm)

GarciaRodriguez et al. (2014)

Flow rate 0.8 mL min21

15
20 min, 20%
80% B

C18

Gradient elution

0
3 min, 20%
40% B

Chaharlangi et al. (2015)

150 mm 3 4.6 i.d., 5 μm

A: H2O, B: ACN

3
8 min, 40%
50% B

DAD (440, 330, 250 nm)

Flow rate 1 mL min21

8
12 min, 50%
50% B

DAD (440, 330, 250 nm)

GarciaRodriguez et al. (2017)

12
15 min, 50%
80% B Development of a HPLC-DAD method for the determination of the safranal content

Phenomenex Luna C18

Gradient elution

0
5 min, 80%
20% B

150 mm 3 4.6 i.d., 5 μm

A: H2O, B: ACN

5
15 min, 20%
80% B

Flow rate 0.8 mL min21

15
20 min, 20%
80% B (Continued )

276

SECTION | IV Saffron processing

TABLE 16.6 (Continued) Aim of the study

Chromatographic column

Mobile phase

Elution conditions

Detector

Reference

Development of a method for the determination of saffron compounds using HPLC-DAD

KNAUER Eurospher C18 250 mm 3 16, 10 μm

Gradient elution

0
5 min, 10%
90% B

Kabiri et al. (2017)

A: H2O, B: ACN

5
15 min, 80%
20% B

DAD (440, 308, 250 nm)

Flow rate 0.8 mL min21

15
20 min, 80%
20% B

Kinetex C18

Gradient elution

0
1 min, 95%
5% B

Moras et al. (2018)

150 mm 3 2.1 i.d., 2.6 μm

A: H2O (0.01%, v/v, formic acid), B: ACN

1
9 min, 60%
40% B

DAD (440, 330, 310, 250 nm)

Quality assessment of saffron using UHPLC-DAD-MS and detection of adulteration with gardenia fruit extract

Flow rate 0.6 mL min21

9
15 min, 0%
100% B 15
17 min, 0%
100% B 17
17.1 min, 95%
5% B

Development of an UHPLC-MS/MS procedure to determine crocins as a marker of quality

Quantitative HPLC-based metabolomics of Iranian saffron samples

Kinetex C18

Gradient elution

0
0.1 min, 95%
5% B

100 mm 3 2.1 i.d, 1.7 μm

A: H2O (5 mM formic acid), B: ACN (5 mM formic acid)

0.1
13 min, 1%
99% B

MS (ESI, positive ion mode)

MS (ESI, negative ion mode)

Rocchi et al. (2018)

DAD (440, 308, 250 nm)

Vahedi et al. (2018)

13
17 min, 1%
99% B

Flow rate 0.3 mL min21

17
20 min, 95%
5% B

KNAUER Eurospher C18

Gradient elution

0
5 min, 10%
90% B

250 mm 3 4.6 i.d., 5 μm

A: H2O, B: ACN

5
25 min, 80%
20% B

Flow rate 1 mL min21

25
30 min, 80%
20% B

FIGURE 16.4 Representative RP-HPLC-DAD profile of an aqueous saffron extract at 440 nm; Insert, Representative RP-HPLC-DAD profile at 250 nm. Peak assignment as described in Table 16.3. Data from the saffron sample collection of LFCT.

Bioactive ingredients of saffron: extraction, analysis, applications Chapter | 16

277

TABLE 16.7 UV-Vis spectra, λmax values and major ions (m/z) of individual apocarotenoids of an aqueous saffron extract at 440 and 250 nm.a Peak

UV-Vis spectra

1

4.26 Min Lambda Max

Major ions (m/z)

Nomenclatureb

247

375 [M-H 1 HCOOH]2

Picrocrocin

442, 461

329 [{(M 1 Na 1 H)-G}G]1,

trans-di-(β-Dgentiobiosyl) crocetin ester (trans 2 4-GG)

300

247

300

λmax

200

100

100 409

344

200

0

0 250

300

350

400

450

500

550

nm

2

5.33 Min Lambda Max 1500 461

442

1500

1000

500

500

260

1000

508 [(M 1 K)/2]1, 1015 [M 1 K]1

0

0 250

300

350

450

400

500

550

nm

3

5.80 Min Lambda Max


c

trans-(β-Dneapolitanosyl)-(β-Dglucosyl) crocetin ester (trans 2 4-ng)

441,462

329 [{(M 1 Na 1 H)-g}G]1,

trans-(β-D-gentiobiosyl)(β-D-glucosyl) crocetin ester (trans 2 3-Gg)

40 441

239

40

441,461

20

20

0

0 250

300

350

400

450

500

550

nm

4 500

500

462

441

6.11 Min Lambda Max

427 [(M 1 K)/2]1, 837 [M 1 Na]1,

250

259

250

0

0 250

300

350

400

450

500

550

nm

40

243

438

6.98 Min Lambda Max

20

438,460


c

trans-di-(β-D-glucosyl) crocetin ester (trans 2 2gg)

20

226

5

40

0

0 250

300

350

400

450

500

550

nm

(Continued )

278

SECTION | IV Saffron processing

TABLE 16.7 (Continued) Peak

UV-Vis spectra

6

100

100

7.86 Min Lambda Max

Major ions (m/z)

Nomenclatureb

325, 435,456

329 [{(M 1 Na 1 H)-G}G]1,

cis-di-(β-D-gentiobiosyl) crocetin ester (cis 2 4GG)

508 [(M 1 K)/2]1,

50

245

325

50

λmax

1015 [M 1 K]1 0

435

0

300

250

350

400

450

500

550

nm

7

25

322

246

25

434

8.64 Min Lambda Max

322, 434,457

trans-(β-D-gentiobiosyl)(β-D-glucosyl) crocetin ester (cis 2 3-Gg)

837 [M 1 Na]1

0

0

329 [{(M 1 Na 1 H)-g}G]1,

-25

-25

250

300

400

350

500

450

nm 8.88 Min Lambda Max

200 435

200

435,457


c

457

8

trans-mono-(β-Dgentiobiosyl) crocetin ester (trans 2 2-G)

100 247

100

0

0

250

300

350

400

450

500

550

nm

a

Chromatographic conditions as described in detail by Kyriakoudi et al. (2012). G, gentiobiose; g, glucose; n, neapolitanose (nomenclature is in accordance with Carmona et al., 2006b). Not detected under the applied chromatographic conditions.

b c

well separated using common C18 columns with particle sizes greater than 3 μm. Taking into consideration all the above, UHPLC is a promising tool in saffron analysis. The adoption of a UHPLC rather than a HPLC protocol for the analysis of saffron apocarotenoids should be considered in a forthcoming revision of the ISO 3632-2 standard. The main weakness regarding quantification of saffron apocarotenoids is the lack of commercially available standards or their high price. Regarding crocins, commercially available standards are either of uncertified purity (microscopy grade) or expensive and available only through a small number of companies [LGC Standards (United Kingdom), ALB Materials Inc (United States), Tauto Biotech Co. Ltd. (China), ChemFaces (China), Fluka (Germany)]. This renders their determination with accuracy very difficult. To overcome this limitation, quantification of crocetin sugar esters after separation was attempted using (1) artificial colorants (e.g., 4-nitroaniline) as internal standards (Lozano et al., 2000) and (2) values of the molecular coefficient absorbance (ε) of trans- and cis- crocetin esters at 440 nm (89,000 and 63,350, respectively) (Alonso et al., 2001; Caballero-Ortega et al., 2007; Carmona et al., 2006a). In this context, a mathematical equation (Eq. 16.2) based on the peak area of each crocetin sugar ester with respect to the total peak area at 440 nm and also on the molecular coefficient absorbance of each compound has been suggested by Sa´nchez et al. (2008) for the quantification of total and individual crocetin esters.

Bioactive ingredients of saffron: extraction, analysis, applications Chapter | 16

% of crocetin esteri on dry basis 5

½MWi 3 ðE1% 440 nm Þ 3 Ai  3ε 10

279

(16.2)

where MWi is the molecular weight of the crocetin esteri, E1%440 nm is the coloring strength, Ai is the percentage peak area of the crocetin esteri at 440 nm, and ε is the molecular coefficient absorbance value. The same research group (Del Campo et al., 2010) proposed a similar mathematical equation (Eq. 16.3) for the quantification of picrocrocin some years later. % of picrocrocin on dry basis 5

½MWi 3 ðE1% 250 nm Þ 3 Ai  3ε 10

(16.3)

where Mwi stands for the molecular weight of the picrocrocin, E1%250 nm is the flavor strength, Ai is the percentage peak area of picrocrocin at 250 nm, and ε is the molecular coefficient absorbance value of picrocrocin (10,100). Some efforts to use laboratory isolated standards for the quantification of total and individual crocins as well as picrocrocin have been described in the literature. In particular, Sa´nchez et al. (2009) isolated picrocrocin with column chromatography via a C18 adsorbent. The chromatographic purity of the isolated picrocrocin was calculated as the percent of the total peak area at 250 nm and was found to be 96%. Kyriakoudi et al. (2012) quantified the total and the two main individual crocins of saffron (i.e., trans 2 4-GG and trans 2 3-Gg crocetin esters) after their separation with the aid of an appropriate calibration curve of in-house isolated trans 2 4-GG crocetin ester. Its isolation was carried out by semipreparative RP-HPLC, its identity was confirmed by LC-ESI-MS analysis and by NMR spectroscopy, and its purity was found to be 98%. The isolation of picrocrocin using thin layer chromatography was reported by Cossignani et al. (2014). Koulakiotis et al. (2015) reported the development of an HPLC-DAD method for the quantification of trans 2 4-GG and trans 2 3-Gg crocetin ester as well as their cis-isomers and trans 2 2-gg crocetin ester. The respective compounds were isolated using semipreparative HPLC, identified by UPLC-ESI-MS and MS/MS analysis, and their purity was found to be 98%. In addition, Tong et al. (2015b) also used in-house isolated standards for the quantification of trans 2 4-GG and trans 2 3-Gg crocetin esters, their cis-isomers, and trans 2 2-G crocetin ester with purity of .95%. Kabiri et al. (2017) also used semipreparative HPLC to isolate trans 2 4-GG and picrocrocin. The purity of the isolated compounds was found to be 97.2% and 91.1%, respectively. Moreover, a molecularly imprinted polymer using gentiobiose as a template (G-MIP) has been reported as the sorbent in a solid-phase extraction method for the selective extraction of trans 2 4-GG from a methanolic saffron extract (Mohajeri et al., 2010). The authors observed that the GMIP had significantly higher affinity to trans 2 4-GG than other compounds and allowed its selective extraction with a recovery of B84%. For the quantification of safranal with HPLC, commercially available standards are usually used (e.g., Garcia-Rodriguez et al., 2017; Moras et al., 2018; Vahedi et al., 2018).

16.4.3 Gas chromatography-mass spectrometry (GC-MS) Besides HPLC, GC coupled to MS has also been used over the years for the determination of safranal content, taking into consideration its volatile nature. Modifications regarding the characteristics of the chromatographic column, the oven temperature program, as well as the sample preparation procedures are numerous. An overview of the gas chromatographic conditions for the analysis of safranal and other volatiles in saffron up to today is given in Table 16.8. In particular, capillary chromatographic columns are used for the analysis of safranal and other saffron volatiles, with lengths that range from 30 up to 60 m, internal diameter from 0.22 to 0.32 mm, and particle size from 0.25 to 0.5 μm. Various techniques have been reported in the literature for the extraction or isolation of volatile compounds from saffron including HD, MSDE, TD, SPME, UAE, etc. Regarding safranal, as in the case of HPLC, its quantification is typically carried out with the aid of appropriate calibration curves of commercially available standard of high purity (e.g., Anastasaki et al., 2009; Kanakis et al., 2004; Liu et al., 2018).

16.4.4 Electronic nose technique Electronic nose (e-nose) is a rapid and powerful technique that does not require any special sample preparation and ´ allows the determination of the volatile profile of a product as a whole (Gliszczy´nska-Swigło and Chmielewski, 2017). In particular, e-noses are devices that consist of an array of various types of sensors, which are able to mimic the sense of smell. The sensors are treated with various odor-sensitive chemical compounds, each one of them yielding a specific fingerprint, the so called smellprint. These patterns are then used to create a database that can be used in order to identify unknown odors (Peris and Escuder-Gilabert, 2016). Taking into consideration that the volatile profile of a product

280

SECTION | IV Saffron processing

TABLE 16.8 Overview of the extraction techniques and the chromatographic conditions for the analysis of safranal and other volatiles in saffron using GC-MS. Aim of the study

Extraction technique

Chromatographic column

Chromatographic conditions

Reference

Qualitative determination of volatile compounds of saffron and quantitative determination of safranal.

Microsimultaneous hydrodistillation extraction, ultrasound-assisted extraction

HP-5 ms capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness)

Carrier gas: Helium

Kanakis et al. (2004)

Flow rate: 1 mL min21 Column temperature: 50 C (3 min), 3 C/ min to 180 C, 15 C/ min to 250 C (5 min) Injection volume: 1 μL (splitless mode)

Analysis of the volatile profile of saffron

Thermal desorption

Study of the seasonal variation of saffron based on aroma constituents

Solid-phase microextraction

BP21 capillary column (50 m, 0.22 mm i.d., 0.25 μm film thickness) ZB-5 MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness)

Carrier gas: Helium Column temperature: 100 C (5 min), 18 C/ min 210 C (15 min) Carrier gas: Helium Flow rate: 0.8 mL min21

Carmona et al. (2006a)

D’Auria et al. (2006)

Column temperature: 40 C (2 min), 8 C/ min to 250 C (splitless mode)

Quantitative structureretention relationship study of saffron aroma compounds based on the projection pursuit regression method

Solid-phase microextraction

Geographical differentiation of saffron

Ultrasound-assisted extraction

ZB-5 MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness)

HP-5 ms capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness)

Carrier gas: Helium Flow rate: 0.8 mL min21

Du et al. (2008)

Column temperature: 40 C (2 min), 8 C/ min to 250 C (splitless mode) Carrier gas: Helium Flow rate: 1 mL min21

Anastasaki et al. (2009)

Column temperature: 50 C (3 min), 3 C/ min to 180 C, 15 C/ min to 250 C (5 min) Injection volume: 1 μL (splitless mode)

Characterization of volatile compounds of Iranian saffron

Ultrasound-assisted extraction

HP-5 ms fused silica capillary column (60 m, 0.25 mm i.d., 0.25 μm film thickness)

Carrier gas: Helium Flow rate: 1 mL min21

JalaliHeravi et al. (2009)

Column temperature: 60 C (1 min), 5 C/ min to 200 C (1 min), 20 C/min to 280 C (21 min) Injection volume: 1 μL (split ratio 1:5) (Continued )

Bioactive ingredients of saffron: extraction, analysis, applications Chapter | 16

281

TABLE 16.8 (Continued) Aim of the study

Extraction technique

Chromatographic column

Chromatographic conditions

Reference

Determination of safranal for the quality control of saffron

Ultrasound-assisted extraction

HP-5 ms capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness)

Carrier gas: Helium

Maggi et al. (2011)

Flow rate: 1 mL min21 Column temperature: 50 C (3 min), 3 C/ min to 180 C, 15 C/ min to 250 C, 250 C (5 min) Injection volume: 1 μL (splitless mode)

Determination of aroma compounds of Iranian saffron

Liquid
liquid extraction (LLE), solid-phase extraction (SPE), simultaneous distillation/ extraction (SDE), solventassisted flavor extraction (SAFE)

DB-Wax capillary column (30 m 3 0.25 mm i. d., 0.5 μm thickness)

Carrier gas: Helium Flow rate: 1.5 mL min21

Amanpour et al. (2015)

Column temperature: 5 C/min to 200 C, 8 C/min to 260 C, 260 C (5 min) Injection volume: 3 μL (splitless mode)

Geographical discrimination and commercial categorization of saffron

Steam distillation

Optima-5 capillary column (30 m 3 0.32 mm i. d., 0.25 μm)

Carrier gas: Helium Flow rate: 1.5 mL min21

Liu et al. (2018)

Column temperature: 5 C/min from 80 C to 150 C, 25 C/min to 250 C (split ratio 4:1)

is specific and allows its discrimination from adulterated ones, e-nose is a very promising tool not only for monitoring ´ food quality but authenticity as well (Gliszczy´nska-Swigło and Chmielewski, 2017). The use of e-nose has been already reported for the quality assessment or authenticity control of various products such as coffee (e.g., Buratti et al., 2015; Severini et al., 2015), milk (e.g., Yu et al., 2007), olive oil (e.g., Cerrato Oliveros et al., 2002), honey (e.g., Benedetti et al., 2004; Dymerski et al., 2014), tea (e.g., Bhattacharyya et al., 2008; Dutta et al., 2003) etc. However, the applications of the e-nose for saffron samples are limited. Carmona et al. (2006a) were the first to use an e-nose based on 27 metal oxide semiconductor (MOS) gas sensors coupled to principal component analysis (PCA) in order to discriminate saffron samples with different geographic origins (i.e., Iran, Morocco, Greece, Spain). In this way, discrimination with 90% confidence was achieved. An e-nose based on 6 MOS gas sensors coupled to PCA has also been used by Heidarbeigi et al. (2015) to detect adulteration in saffron samples. In particular, the authors examined the volatile fingerprint of pure saffron, saffron with yellow stamens, and safflower. This e-nose system allowed discrimination among pure and adulterated saffron samples at adulteration above 10%. Kiani et al. (2016) reported the use of a portable enose based on 10 MOS gas sensors coupled to a multilayer perceptron artificial neural network for the discrimination of saffron samples from various regions of Iran. The authors suggested that the developed system is inexpensive, nondestructive, and allows discrimination with a 100% success rate. The same research group (Kiani et al., 2017) used a similar e-nose system based on gas sensors for the quantitative characterization of safranal and other saffron volatile compounds. For the prediction of the E1%330 nm values, an unsupervised pattern recognition model was used. The authors suggested that the e-nose is a nondestructive technique appropriate for the analysis of safranal content in saffron samples without the need for prior extraction.

282

SECTION | IV Saffron processing

16.5 Applications of saffron bioactive ingredients: from prehistory up to 21st century Saffron has been used for various purposes such as spice, medicine, dye, and perfume by different nations throughout history. Its particular significance is illustrated in the famous fresco fragments of the Bronze Age found in Thira (Santorini, Greece) that depict a female goddess figure surrounded by a young girl and a monkey in a landscape filled with Crocus plants. More specifically, the young girl seems to gather crocus flowers in a basket, whereas the monkey extends some stigmas toward the goddess. Moreover, the lower part of the fresco shows a young woman who seems to use saffron for the treatment of her bleeding foot. Analysis of the fresco (Ferrence and Bendersky, 2004) suggests that not only a description of the saffron production line is illustrated, but also its medicinal and healing properties are exhibited. The first report regarding the use of saffron as an aid in dyspnea, urination, menstrual disorders, and childbirth is dated to the Bronze Age. Subsequently, Hippocrates, Erasistratus, Diokles, and Dioscorides used saffron in different historical periods for various medicinal purposes such as treatment of eye diseases (e.g., cataract) and toothache, as an aphrodisiac and emmenagogue, as well as for its styptic and soothing properties. The use of saffron for cosmetic purposes is also reported since ancient times. Saffron was also used as a dye for expensive royal fabrics such as silk, cotton, and wool. Up to today, saffron is highly valued in the food industry for the unique color, taste, and aroma that it imparts to food preparations. It is used as a spice, either in filaments or in powder form, in many traditional European dishes such as the Spanish paella, the French soup bouillabaisse, the risotto alla Milanese, as well as in many Greek bakery products found in the Cyclades and Dodecanese islands. Saffron consumption is more common in Iran, where B90% of the world’s total annual saffron production originates from. Moreover, countries of the Arabian Peninsula as well as Egypt, consume high quantities of saffron throughout the year even though most of them do not produce this spice but import it either in bulk form or packed in small quantities. Apart from its use in food preparations, saffron is used in Arabic coffee, the most popular and common drink in these countries. Saffron is also used in the preparation of distinct bakery products meant to be consumed during a certain time period [e.g., “saffron buns” that are traditionally prepared on St. Lucia Day celebrated during Christmas (13th of December) in Sweden or Labrokouloura that are consumed during the Easter period in Astypalaia (Cyclades, Greece)] (Kyriakoudi and Tsimidou, 2018a; Ordoudi and Tsimidou, 2004). In the following sections, emphasis is given only on applications of saffron for which scientific evidence exists.

16.5.1 Food industry Dairy products: Due to its sensorial attributes, saffron finds applications in dairy products and more specifically in certain types of cheese such as the Piacentinu Ennese, a sheep’s milk hard Protected Destination of Origin (PDO) cheese from Sicily, the semihard cheese Pecorino allo Zafferano from Italy made from pasteurized sheep’s milk, as well as an Austrian cow’s milk cheese called Lu¨neberg. Effects of the concentration of saffron on the chemical, sensorial, textural, and microbiological characteristics of a pressed sheep’s milk cheese during ripening have been examined by Lico´n et al. (2012). The authors found that cheeses containing saffron were more yellow, firmer, more elastic, and microbiologically more stable compared to the ones without saffron. The research group of Polysiou (Aktypis et al., 2018) supplemented a fresh ovine cheese with saffron and examined its microbiological, physicochemical, antioxidant, color, and sensory characteristics. The authors found that even though no significant changes in the physicochemical properties of the cheese were observed, its antimicrobial and antioxidant activity was enhanced due to the presence of saffron. Cereal products: Saffron has been added to the formulation of fresh pasta, which after cooking was evaluated in terms of textural, physicochemical, and sensory properties (Armellini et al., 2018). The authors found that the addition of saffron increased the acceptability of the saffron enriched pasta in terms of visual aspect, color, aroma, taste, chewiness, hardness, gumminess, and overall acceptability. Moreover, the authors claim that the use of saffron enhanced the antioxidant activity of the final product based on the DPPH and ABTS assays. Desserts: Almodo´var et al. (2018) used a commercial saffron extract with standardized (by HPLC) amount of crocins to prepare two cold saffron flavored desserts, namely white chocolate soup with yogurt and saffron as well as a cheese cake with orange jam and saffron. The authors suggested that the use of the commercial standardized saffron extract allowed more precise dosage control resulting in increased consumer acceptability compared to the use of saffron stigmas. Alcoholic and nonalcoholic beverages: Extracts of saffron are also used in alcoholic and nonalcoholic beverages such as Strega, Benedictine, vermouth, and other bitter drinks, as well as in herbal teas (e.g., http://www.krocuskozanis.com/). Saffron bitterness is a limiting factor for consumer acceptance in nonalcoholic beverages (Chrysanthou et al., 2016).

Bioactive ingredients of saffron: extraction, analysis, applications Chapter | 16

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Ordoudi et al. (2015) examined a variety of infusions prepared from commercially available herbal tea blends with saffron in terms of chemical characterization as well as determination of the bioaccessibility of crocins. Results showed that the copresence of strong phenolic antioxidants from the other herbs enhanced the bioaccessibility of crocins, which was determined using an in vitro gastrointestinal digestion model. The authors suggested that the findings of this study could be exploited in the design of novel saffron-based functional beverages. It is worth mentioning that to the best of our knowledge, information concerning the bioaccessibility and bioavailability of saffron apocarotenoids is rather limited (Asai et al., 2005; Kyriakoudi et al., 2013, 2015a; Lautenschla¨ger et al., 2015; Ordoudi et al., 2015). Such data are of particular importance in order to establish oral intake limits and recommendations, since only a certain amount of a bioactive compound is actually absorbed in the blood circulation and reaches the target tissues after ingestion. In general, a critical factor for the use of saffron apocarotenoids not only in food but also in pharmaceutical and cosmetics applications, as shown below, is their stability. Being unsaturated compounds, saffron apocarotenoids are prone to degradation. Their stability has been examined under the influence of various parameters such as temperature, pH, water activity, oxygen, and light (Alonso et al., 1990; Tsimidou and Biliaderis, 1997; Tsimidou and Tsatsaroni, 1993). There is a growing interest of the scientific community in the protection of these precious apocarotenoids via encapsulation. In particular, saffron extracts as well as isolated crocetin sugar esters and crocetin have so far been encapsulated in different matrices such as polyvinylpyrrolidone, pullulan, maltodextrin, gum Arabic, deoxycholic acid, etc. using various techniques such as freeze drying (Jafari et al., 2016; Chranioti et al., 2015; Selim et al., 2000), spray drying (Esfanjani et al., 2015; Kyriakoudi and Tsimidou, 2018c; Rajabi et al., 2015; Zhou et al., 2013), and inclusion complexation (Kyriakoudi and Tsimidou, 2015).

16.5.2 Pharmaceutical industry Even though saffron is mainly used in the food industry as a spice, a variety of pharmacological properties (e.g., anticarcinogenic, antioxidant, neuroprotective, cardioprotective, antiinflammatory, antidiabetic, etc.) have been attributed either to saffron extracts or to its major apocarotenoids. This is the reason why saffron has been characterized as a functional spice (Kyriakoudi et al., 2015b). There are numerous book chapters and review articles that summarize or critically focus on its most important biological actions, which are examined with either in vivo, ex vivo, or in vitro studies (Alavizadeh and Hosseinzadeh, 2014; Bhandari, 2015; Bukhari et al., 2018; Finley and Gao, 2017; Giaccio, 2004; Leone et al., 2018; Pitsikas, 2016; Singla and Bhat, 2011; Ulbricht et al., 2011). From the wide spectrum of biological actions of saffron, in the present work, emphasis is given to the most promising applications against critical chronic diseases such as Alzheimer’s disease, opthalmological diseases, as well as depression and anxiety. Alzheimer’s disease: Many researchers have focused on the use of saffron extracts or its bioactive apocarotenoids for the potential treatment of Alzheimer’s disease, which is the most common form of dementia among people over the age of 65 worldwide (Shahi et al., 2016). Alzheimer’s is an irreversible progressive neurodegenerative disease characterized by behavioral disturbances, cognitive deterioration, and functional disability (Adalier and Parker, 2016). In particular, consumption of 30 mg day21 saffron in two doses of 15 mg each for a period of 22 weeks by 54 adults elicited similar effects as consumption of 10 mg day21 donepezil (5 mg twice per day) in treatment of mild to moderate Alzheimer’s disease (Akhondzadeh et al., 2010a). In another study, the same research group (Akhondzadeh et al., 2010b) carried out a randomized, double-blind, placebo-controlled trial and showed that consumption of 30 mg day21 (15 mg twice/day) saffron for 16 weeks by 46 patients with probable Alzheimer’s disease resulted in better outcomes compared to placebo. Based on these results, the authors suggested that saffron is safe and effective in the treatment of mild to moderate Alzheimer’s disease, at least in the short-term. Another randomized, double-blind clinical trial was conducted in 68 patients with moderate to severe Alzheimer’s disease (Farokhnia et al., 2014). Patients received saffron extract (30 mg day21) or memantine (20 mg day21) for 12 months. The study showed that the 1-year saffron administration exhibited comparable results to memantine in terms of reducing cognitive decline in patients with moderate to severe Alzheimer’s disease. Tiribuzi et al. (2017) investigated in vitro the ability of trans-crocetin to restore the reduced ability of monocytes of Alzheimer’s disease patients to degrade amyloid-β(1
42) (Aβ42). This protein is the main component of the amyloid plaques found in the brains of Alzheimer patients. Trans-crocetin (5 μM) was found to enhance Aβ42 degradation in monocytes after incubation for 24 hours. The authors suggested that trans-crocetin could be potentially used as a new antiamyloid agent. In another study (Finley and Gao, 2017), the multifunctional protective activities of crocins in brain cells as well as its potential to improve learning and memory are presented. Based on scientific evidence, the authors suggested that crocins could be a promising agent for the prevention or treatment of Alzheimer’s disease.

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Visual impairment: Incubation of photoreceptors in retinal cell cultures with trans 2 4-GG (30 μM) was found to increase their viability against blue and white light induced death (light stress) (Laabich et al., 2006). Based on these results, the authors suggested that trans 2 4-GG as well as other crocins could be used as possible therapeutic agents for degenerative diseases of the retina. In a similar in vivo study, crocetin (100 mg kg21) was found to inhibit photoreceptor degeneration and retinal dysfunction, indicating that crocetin has protective effects against retinal damage (Yamauchi et al., 2011). In an in vivo study in rats, 8 weeks of administration of crocins by intraperitoneal injection showed beneficial effects on prevention of diabetic cataracts (Bahmani et al., 2016). This effect was attributed by the authors to fact that crocins, as antioxidant and hypoglycemic agents, decreased protein glycation and prevented cataract formation. Depression and anxiety: Depression and anxiety are the most common phycological disorders. Saffron has been reported to relieve their symptoms and even treat them. In particular, Tabeshpour et al. (2017) examined the effects of saffron on postpartum depression with a double-blind and placebo-controlled clinical study. In particular, 60 breastfeeding mothers with mild to moderate depression were randomly divided into two groups. Half women received 30 mg day21 in the form of a capsule and the other half received an equivalent dose of placebo for 8 weeks. The results of this study showed that saffron was more effective than the placebo in treating postpartum depression in new mothers. The authors suggested that saffron could be used as an alternative medication for reduction of the symptoms of postpartum depression. Another double-blind and placebo-controlled clinical trial examined the effects of an alcoholic saffron extract on mild-to-moderate comorbid depression-anxiety and sleep quality of 54 patients (Milajerdi et al., 2018). Saffron (30 mg day21) or placebo capsules of similar dose were administered to the patients for 8 weeks. The authors found that saffron relieved the symptoms of mild to moderate depression, anxiety, and sleep disturbances. Lopresti and Drummond (2014) carried out a randomized, double-blind, placebo-controlled trial to examine the effects of saffron on anxiety and depressive symptoms of 80 patients ages of 12
16. Half of them were given a tablet containing 14 mg of a standardized saffron extract, and the other half was given a placebo tablet. The authors suggested that after 8 weeks saffron was found to relieve the anxiety and depressive systems.

16.5.3 Cosmetics and other sectors Taking into account all of the pharmacological actions of saffron, a renewed interest in its exploitation in various types of cosmetics has come about. However, to the best of our knowledge, the relevant research articles found in the literature are still limited. More specifically, Fekrat (2004) reported the use of an ethanolic saffron extract in sun protection creams, body lotions, shampoos, hair care products, moisturizing creams, as well as liquid soaps. Some years later Vyas et al. (2010) used a concentrated saffron dry extract as an ingredient in a cream, a lotion, and a face powder. The properties of these formulations were examined with patch tests on various subjects ages 18
28. The results showed increased shining and lightening of the skin compared to a control product. The authors attributed this observation to the presence of crocins. Taking into consideration that the process of ageing involves not only reactive oxygen species but also inflammatory mediators, saffron could be potentially used against ageing. Toward this end, a group of researchers from L’Ore´al Company (Fagot et al., 2018) examined the antioxidant and antiinflammatory effects of crocin on normal human keratinocytes and fibroblasts in vitro. The researchers found that the production of proinflammatory mediators (e.g., IL-6, IL-8, PGE2, TNFα) is inhibited in the presence of trans 2 4-GG crocetin ester, whereas antioxidant defense is increased. Based on their findings, the authors suggested that trans 2 4-GG crocetin ester could be exploited as a promising skin ageing prevention cosmetic agent. The UV protective effects of saffron have been also highlighted. In particular, Zarkogianni and Nikolaidis (2016) prepared oil-in-water emulsions containing saffron, which were examined for their antisolar activity by measuring their sun protection factor (SPF). The authors suggested that saffron could be used as a natural UV-absorbing agent in sunscreen products. In the same context, Ntohogian et al. (2018) prepared sunscreen oil-in-water emulsions based on chitosan nanoparticles with saffron. The authors concluded that the prepared chitosan-saffron nanoparticles showed thermal and color stability for up to 90 days as well as minimal sunscreen protection (SPF 2.15
4.85) compared to blank emulsion (SPF 5 1.00). Even though there are only a few relevant published research articles, numerous cosmetic products containing saffron are already commercially available [e.g., a serum that claims to correct all signs of aging (https://www.korresusa. com/skincare/golden-krocus-ageless-saffron-elixir) and face creams to promote skin glow and nourishment (https:// www.stylecraze.com/reviews/lever-ayush-natural-fairness-saffron-face-cream/#gref, https://www.alibaba.com/productdetail/Saffron-Whitening-Cream-100g-Paraben-Free_50018430835.html)]. Applications regarding the use of saffron as a natural dye for textiles have also been reported in the literature. However, such reports are limited due to saffron’s high price. In particular, Tsatsaroni and Eleftheriadis (1994)

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used aqueous saffron extracts to dye cotton and wool, which constitute suitable substrates for natural dyes. Dyeing of cotton and wool by a methanolic saffron extract has also been reported by Liakopoulou-Kyriakides et al. (1998) after enzymatic treatment with α-amylase, which is said to minimize wool shrinkage and improve the dyeing properties.

16.6

Conclusion

Evidence in this chapter demonstrates the continuing interest of scientists, mainly from saffron producing countries, to update and modernize means of extraction and analysis of saffron bioactive compounds. The interest in bioactivity studies and applications is rather universal, though once again most of the studies come from scientists in saffron producing countries. Scientific results support the multidisciplinary interest in the exploitation of the properties and uses of this most precious commodity.

Acknowledgment Seid Mahdi Jafari appreciates the support of his saffron project by The World Academy of Sciences (TWAS)- a UNESCO sub-organization under grant code of 14-309 RG/MSN/AS_C- UNESCO FR: 3240283416 (2015). MZT and AK acknowledge support of this work by the project “Upgrading the Plant Capital (PlantUp)” (MIS 5002803), which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Enterpreneurship and Innovation” (NSRF 2014
2020) and cofinanced by Greece and the European Union (European Regional Development Fund).

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646.

Chapter 17

Dehydration of saffron stigmas Arash Koocheki Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 17.1 17.2 17.3 17.4 17.5 17.6

Introduction Drying methods Traditional methods Artificial drying methods Hybrid photovoltaic
thermal solar dryer Infrared thin-layer drying

17.1

291 292 292 292 293 294

17.7 Freeze drying 17.8 Microwave drying 17.9 Effects of drying on color, aroma, and taste 17.10 Conclusion References

295 296 296 297 298

Introduction

Most foods are highly perishable, and therefore awareness of shelf-life is vital if products are to be stored in a warehouse or by the consumer for substantial periods. Drying is a traditional and useful method for keeping solid foods safe for long periods of time. When food products are exposed to drying conditions, the native physical state of the food product is altered, leading to changes in the quality and safety of the food product. Drying involves removal of excess water from the food matrix until a “safe” moisture level is achieved, at which minimal or no physical, chemical, or microbiological reactions occur. Moisture content in saffron is a critical parameter for preservation of its characteristics (Alonso et al., 1993). To keep saffron preserved for a longer period of time, it should be dried. Postharvest processing, such as drying methods and storage conditions, determines the stability of saffron, which directly affects the market value of the product. Drying operations therefore need to be precisely controlled and optimized. This is necessary to produce a high-quality product that has the highest level of color and flavor, while maintaining microbiological safety. During the dehydration process, the stigmas lose B80% of their weight. A lower moisture content, at least below 12%, maintains the quality of saffron for a longer time (International Standard ISO 3632). Drying must be done in a proper way to achieve the right moisture content level. If the stigmas are not dried soon after picking, they are attacked by molds. On the other hand, if saffron is dried too much, it breaks easily, turns into powder, and loses weight to below the trade requirements. The equilibrium moisture content is dependent on the temperature and relative humidity of the environment as well as the species, variety, and maturity of the plant (Iglesias and Chirife, 1982; Rahman, 1995). The dehydration process not only plays an important role in the preservation of saffron, but it is a critical step in development of the principal substance responsible for saffron’s aroma, safranal (Del Campo et al., 2010). Fresh stigmas are not able to impart their organoleptic characteristics to food. A dehydration treatment, which is necessary to convert Crocus sativus L. stigmas into saffron spice, brings about the physical, biochemical, and chemical changes necessary to achieve the desired attributes of saffron (Carmona et al., 2005). The most obvious characteristic of saffron is its deep red color. The high gloss of fresh stigmas becomes dull upon drying, and a strong yellow extract passes into water upon wetting the dried stigmas. Water activity (aw) is an important parameter to assess the shelf-life of foods. Many desired and undesired physical changes in food can be correlated to the water activity of the system. Furthermore, water activity can have a major

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00017-4 © 2020 Elsevier Inc. All rights reserved.

291

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impact on the color, taste, and aroma of foods. The loss of saffron color is reduced at medium water activity of 0.43
0.53 (Tsimidou and Biliaderis, 1997) and the development of safranal is promoted.

17.2

Drying methods

Drying by any method requires considerable skill to ensure a high-quality spice. The main attributes that determine the market value and quality of saffron are color, aroma, and taste. The compounds responsible for these attributes are crocins, safranal, and picrocrocin, respectively. The quantities of these compounds in saffron depends mainly on the method by which the stigmas are dried. The drying process differs from country to country (Ordoudi and Tsimidou, 2004), and the different conditions of drying and aging affect the saffron constituents (Carmona et al., 2005). Low-temperature drying processes are favorable for maintaining high bioactivity of desired biocomponents in the final product. A minimal change in nutritional values is targeted during low-temperature processes. Generally, there are two forms of saffron dehydration: traditional and industrial drying. Three different procedures are employed depending on the temperature used for dehydration: sundried or at room temperature with ventilation (India, Iran, and Morocco), mild temperature (Greece and Italy), and high temperature (Spain) (Carmona et al., 2005).

17.3

Traditional methods

The traditional method of drying saffron varies from country to country and depending on the availability of required equipment (Acar et al., 2011). Natural sun drying is considered to be the most common drying method for saffron worldwide. Solar drying, in sun or in shade, has been used for many years because of its simplicity and low investment cost although it results in a photochemical decrease in color intensity. Natural sun drying takes place outdoors, and the product is directly exposed to sunlight and open to risk of contamination. Therefore, the ultraviolet solar light can degrade or isomerize the carotenoids in saffron. These drying methods are still used in Iran, India, and Morocco for drying of saffron stigmas. In Iran and Morocco, the stigmas are handled very gently and spread in a very thin layer on a large cloth. They are then exposed to the sun for several hours or placed in shade for 7
10 days. Drying is completed before the stigmas break or crumble. Air-dried saffron retains its purplish red color, its fragrance, and its aroma. In India, the stigmas are solar-dried for 3
5 days until their moisture content is reduced to 8%
10%. However, these methods raise some problems such as long drying time and microbial contamination of the dried materials (Gregory et al., 2005).

17.4

Artificial drying methods

Some modern drying methods have replaced the age-old drying method of fine mesh screens held over burning coals (Raines Ward, 1988). Artificial drying methods are carried out at higher temperatures and have been employed in saffron processing in some countries such as Spain, Greece, and Italy (Carmona et al., 2006). High-temperature drying processes require a strong heat supply for the removal of moisture from the food sample. The heat can be supplied in many ways such as microwave, radio frequency, hot gas stream including air, superheated steam, etc. The hot gas stream is the most frequently used heat source for large-scale commercial industries due to its increased availability, easier heat recovery, and cheaper cost compared to other heat sources. For this method the saffron is dried by means of hot air flow or by placing it on a heater. According to Carmona et al. (2005), the highest coloring strength was obtained when saffron was submitted to higher temperatures and shorter times. These findings may be supported by the fact that samples dehydrated at high temperature were more porous than those dehydrated at room temperature. In Italy, the process is carried out by spreading the fresh stigmas on a sieve placed B20 cm above live oak-wood embers. Halfway through the process, the stigmas are turned over to ensure homogeneous drying. The process is considered finished when saffron stigmas retain between 5% and 20% moisture and possess a certain amount of elasticity when pressed between the fingers (Tammaro, 1999). Saffron dried over charcoal maintains its organoleptic qualities better; its purplish red color, its fragrance, and its aroma will be retained (Zanzucchi, 1987). Drying saffron in Spain is accomplished by placing a layer of fresh stigmas less than 2 cm thick on a sieve with a silk bottom. The sieve is placed over the heating source, which can be a gas cooker, live vineshoot charcoal, or, less often, an electric coil. In Castile
La Mancha, in the central Spain the most used source is a gas cooker, followed by embers from kermes oak, or occassionally electrical sources (Alonso et al., 1998). The process is finished when the sample has lost between 85% and 95% of its moisture after being dried at 50 C
80 C for 30
60 minutes. At the

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293

halfway point of the process when 10
15 minutes have passed, the entire mass is turned over with the help of another sieve of the same type, which is again placed over the heating source to finish the drying process. In Greece, when stamens and stigmas are dried together, the stamens’ pollen pollutes and deteriorates the red saffron. It is therefore recommended to separate them before drying. The drying process starts by placing a thin layer of freshly harvested stigmas (4
5 mm) on 40 3 50 cm trays with fine silk screen. These trays are piled on frames with shelves 25
30 cm apart. The frames are then placed in a dark room or in a storage room with controlled temperature, which are then heated with a firewood stove. During the first few hours of the drying process, the temperature is maintained at 20 C and then raised to 30 C
35 C. The drying process is stopped when the moisture content of the product reduces to 10%
11% after 12 hours. If the red (stigmas and styles) and yellow (stamens) components of saffron are still together after drying, they can be separated at this stage. At the same time, all foreign substances (soil, hairs, threads, etc.) are removed from the dried saffron product. The pure dried saffron is kept in hermetically sealed glass vases or tin cans at 5 C
10 C. In spite of some advantages of industrial dryers including achievement of hygienic conditions, quality control, reduction of product loss, and decreased process duration, the energy requirement of drying technology is one of the key problems that should be overcome. The drying process generally consumes large amounts of energy and releases carbon oxides into the environment. Therefore, it is crucial to assure not only good quality of the dried products but also high energy efficiency and low environmental impact (Aghbashlo et al., 2013).

17.5

Hybrid photovoltaic
thermal solar dryer

The energy required for drying, which is mostly for the production hot air, is mainly supplied by fossil fuels. The global demand for fossil fuels and the consequent increase in their prices, the insecurity of their supply, and environmental concerns have resulted in increasing interest in using renewable energies sources such as solar power. Solar energy in its first form (sunlight) can be converted into heat by thermal solar collectors or into electricity by photovoltaic solar cells. A new technology has been developed to combine both types of conversions; this technology is called the solar photovoltaic/thermal collector (PV/T). Hybrid PV/T systems, or PVTs, are systems that convert solar radiation into thermal and electrical energy. These systems combine a solar cell, which converts sunlight into electricity, with a solar thermal collector, which captures the remaining energy and removes waste heat from the PV module (Fig. 17.1). Results of various studies have shown that such hybrid systems are more efficient than both individual photovoltaic and thermal systems.

FIGURE 17.1 Schematic diagram of heat pump-assisted hybrid PV/T solar dryer. From Mortezapour, H., Ghobadian, B., Khoshtaghaza, M.H., Minaei, S., 2014. Drying kinetics and quality characteristics of saffron dried with a heat pump assisted hybrid photovoltaic-thermal solar dryer. J. Agr. Sci. Tech. 16, 33
45.

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In a hybrid PV/T solar dryer, a photovoltaic panel provides the thermal energy required for both moisture removal from the products and electrical power for a fan to circulate air through the dryer. Mortezapour et al. (2012) used a hybrid PV/T system for drying saffron stigmas. To improve the quality of the final product, a heat pump was also added to the system, making it suitable for heat-sensitive materials such as saffron stigma. The sides and back wall of the solar air collector were constructed from wood and insulated by glass wool. A glass sheet was used as the transparent front cover of the solar collector, and a photovoltaic panel was fixed at the middle of the collector sides, with equal distances from the wooden back wall and the top glass cover, to work as the solar irradiance absorber plate. The arrangement of the dryer’s components and the heat pump system, which were connected together by glass wool-insulated round ducts, created a system in which the drying air was circulated in a closed cycle through the evaporator, solar air collector, condenser, auxiliary heater, and drying chamber, respectively. A fresh air valve was deployed to allow ambient fresh air to enter the dryer’s duct and mix with the drying air when temperature and relative humidity of the drying air were more than their desirable set values. Fresh saffron stigma (moisture content of 80% wb) were spread on a tray and placed inside the drying chamber. Results showed that drying time decreased by 62% as the air temperature increased from 40 C to 60 C. Utilizing the heat pump system with the hybrid solar dryer improved drying rate and shortened the drying time by 60%. The color of saffron also improved when drying temperature increased and the heat pump system was applied. Saffron dried with heat pump-assisted hybrid PV/T had great aromatic properties with no significant changes in its bitterness. As a result of these improvements, total drying time and energy consumption are decreased with use of a hybrid PV/T solar dryer. Applying a heat pump with the dryer leads to further reduction in the drying time and energy consumption and an increase in the electrical efficiency of the solar collector. The average total energy consumption is reduced by 33% when the dryer is equipped with a heat pump. Maximum values for electrical and thermal efficiency of the solar collector are 10.8% and 28%, respectively. A maximum dryer efficiency of 72% and a maximum specific moisture extraction rate were obtained at an air flow rate of 0.016 kg s21 and air temperature of 60 C when using the heat pump (Mortezapour et al., 2014).

17.6

Infrared thin-layer drying

Infrared radiation (IR) is an increasingly popular method of supplying heat for drying of moist materials. Infrared drying involves heat transfer by radiation between a hot element and a material at lower temperature that needs to be dried. The peak wavelength of the radiation depends on the temperature of the heated element (Fig. 17.2). IR heating presents advantages such as decreased drying time, high energy efficiency, and lower environmental impact. The energy of radiated waves is transferred from the source to the sample product without heating surrounding air, which leads to higher temperatures in the inner layers of the samples compared to surrounding air and thus a high rate of heat transfer (Celma et al., 2008). Akhondi et al. (2011) investigated the drying of saffron stigma with a laboratory infrared dryer. The influence of temperature on the drying rate of samples at various temperatures (60 C, 70 C, 110 C) was studied. The drying time FIGURE 17.2 Infrared dryer setup schematic. 1, digital balance; 2, infrared heating tube; 3, dimmer; 4, fixed voltage power unit; 5, data logger; 6, hygrometer; 7, k-type thermocouple; 8, t-type thermocouple; 9, samples; 10, inlet cold air; 11, PC. From Ziaforoughi, A., Yousefi, A.R., Razavi, S.M.A., 2016. A comparative modeling study of quince infrared drying and evaluation of quality parameters. Int. J. Food Eng. 12, 1
9.

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FIGURE 17.3 Schematic diagram freeze dryer used for saffron dehydration experiment. From Acar, B., Sadikoglu, H., Doymaz, I., 2015. Freezedrying kinetics and diffusion modeling of saffron (Crocus sativus L.). J. Food Process. Preserv. 39, 142
149.

decreased with an increase in drying air temperature. According to Torki-Harchegani et al. (2017) the total crocin content increased when the IR dryer temperature increased to up to 90 C, but at higher temperatures the amount of crocin slightly decreased. The total safranal content of the samples decreased when the IR drying temperature increased from 60 C to 70 C and then continuously increased to up to 110 C. The amount of picrocrocin also increased as the IR drying temperature increased from 60 C to 100 C. As a result, the maximum values of crocin and safranal were obtained in the samples treated at the highest IR drying temperature.

17.7

Freeze drying

The freeze-drying process can be considered as a drying method for stigmas of the saffron flower (Acar et al., 2015). Freeze drying, also known as lyophilization, is a dehydration process typically used to preserve a perishable material or to make the material more convenient for transport. Freeze drying works by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublime directly from the solid phase to the gas phase (Fig. 17.3). Sublimation is when a solid (ice) changes directly to a vapor without first going through a liquid (water) phase. Controlled freeze drying keeps the product temperature low enough during the process to avoid changes in the dried product’s appearance and characteristics. Therefore, heat-sensitive materials, fine chemicals, biotechnological products, and some pharmaceuticals, which might lose their quality (activity) in conventional evaporative drying, can be safely freeze-dried (Sadikoglu and Liapis, 1997; Sadikoglu et al., 2006). Compared with the other conventional dehydration processes, the highest quality dried product can be obtained by freeze drying. The freeze-drying process is a multistage, relatively slow, and expensive (initial investment and operating costs are high) process. Lyophilized saffron is a new product with a higher content of crocins, a consistent content of native compounds, and minimum water content (about 4%) in comparison to traditional dried saffron. For this reason it could be used as a standard substance to evaluate the quality and water content of saffron powder itself. At the same time the innovative method of lyophilization enables production of saffron with very low moisture content and consequently higher crocins content, longer shelf-life, greater stability, and higher coloring power. In this method, stigmas are placed in the tray of the freeze dryer at a 240 C for 4 hours for complete freezing. The initial temperature of the freeze dryer is 230 C and increases gradually to 5 C without causing any melting or scorching of the stigmas. The drying chamber pressure should be kept at its minimum value (at least it should be well below the ice vapor pressure of the sample being freeze-dried). This increases water vapor mass flux through the pores of the dried material due to sublimation during the primary drying stage (Sadikoglu et al., 2003, 1998). The drying chamber pressure is set at 50 Pa and kept constant during drying. Even though the initial investment and operation costs are high and the drying time is substantially longer than conventional drying methods, freeze drying is considered to be a good method for dehydration of saffron stigmas. The original shape and structure of the sample can be preserved during the freeze-drying process. Freeze-dried saffron not only contains high amounts of safranal and crocin but also has lower moisture compared with traditional and sun-dried saffron. Lower moisture content can prevent fading of the color of the saffron by limiting the degradation of crocin into crocetin during storage. The high cost of freeze drying saffron can be compensated for by keeping the safranal and crocin contents in the final product.

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FIGURE 17.4 A schematic diagram of microwave
convective oven dryer. From Zarein, M., Samadi, S. H., Ghobadian, B., 2015. Investigation of microwave dryer effect on energy efficiency during drying of apple slices. J. Saudi Soc. Agric. Sci. 14, 41
47.

17.8

Microwave drying

The term “microwave” refers to electromagnetic radiation in the frequency range of 300 MHz to 300 GHz with a wavelength of 1 m21 mm (Feng et al., 2012). Microwaves are not forms of heat but rather forms of energy that are manifested as heat through their interaction with materials. It is the propagation of electromagnetic energy through space by means of time-varying electric and magnetic fields (Fig. 17.4). Microwave energy makes it possible to control the drying process more precisely in order to obtain greater yields and better quality products in the shortest possible time. The mechanism for drying with microwave energy is quite different from that of conventional drying. Microwaves initially excite the outer layers of molecules. The inner part of the material is warmed as heat travels from the outer layers inward. Most of the moisture is vaporized before leaving the material. This results in very rapid drying without the need to overheat the atmosphere and perhaps cause case hardening or other surface overheating phenomena. In microwave drying, energy is transferred through the material electromagnetically, not as a thermal heat flux. Therefore, the rate of heating is not limited and the uniformity of heat distribution is greatly improved. This results in a significant reduction in drying time, leading to significantly improved product quality (Schubert and Regie, 2006). In many cases microwaves are at least 50% more efficient than conventional systems, resulting in major cost savings. Microwave drying can be used to improve the chemical profile of saffron in terms of safranal, which is responsible for its aroma. Microwave drying of saffron has a number of quantitative and qualitative advantages over conventional drying methods. In this method heat conductivities or heat transfer coefficients does not play such an important role. Therefore, saffron can be heated in a microwave dryer in a shorter time, with lower drying temperature, and with a more even temperature distribution. In microwave drying, treatments at lower microwave power and longer time benefit the quality of saffron. It only takes 3 minutes at 600 W to dry saffron when moisture is less than 12% and 6 minutes at 400 W. As a result, drying saffron stigma at moderate temperatures in a microwave oven results in saffron with better quality in terms of higher color strength, aroma, and taste.

17.9

Effects of drying on color, aroma, and taste

Postharvest processing such as drying methods and storage conditions determine the stability of saffron, which directly affects the market value of the product. The main active compounds in saffron are: crocins, a group of glycoside derivatives from the carotenoid crocetin; terpenic aldehydes known as safranal; and a glycoside terpenoid, picrocrocin. These compounds are responsible for saffron’s coloring power, bitter taste, and aroma, respectively (Carmona et al., 2006). The quantities of these compounds in saffron are influenced by many factors. The dehydration treatment necessary to convert saffron stigmas into saffron spice is one of the most important factors (Carmona et al., 2006). The effect of temperature and other drying components on spice color and taste remain to be fully determined. There is some evidence that the three types of compounds (crocetin esters, picrocrocin and its related compounds, and volatiles) could be interrelated, as crocetin esters can generate as much picrocrocin as their analogues such as safranal and other volatile compounds (Carmona et al., 2007).

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According to Raina et al. (1996), temperatures lower than 35 C
45 C are required for excessive enzymatic degradation of crocetin esters, the compounds responsible for saffron color. They found that crosin pigment content was highest in saffron dried between 35 C and 55 C either in solar or oven drying. Under these conditions safranal was at its peak value except for the vacuum oven dried samples. On the other hand, using high temperature does not degrade the crocetin ester compounds. In fact, it increases the coloring strength and decreases other crocetin esters such as the trans-crocetin (β-D-glycosyl)-(β-D-gentibiosyl) ester and trans-crocetin (β-D-gentiobiosyl) ester (Carmona et al., 2005). According to Carmona et al. (2005), the highest coloring strength is obtained when saffron is submitted to higher temperatures and lower times. These findings were supported by the fact that samples dehydrated at high temperature were more porous than those dehydrated at room temperature. In addition there is evidence that high temperature promotes the production of compounds responsible for taste and aroma. Maghsoodi et al. (2012) also reported that higher amounts of safranal (aroma) and crocin (color) were obtained at high temperature. However, there was no significant difference between the amounts of picrocrocin at different temperatures. Results by Gregory et al. (2005) showed that a brief (20 minutes) initial period at a relatively high temperature (80 C
92 C) followed by continued drying at a lower temperature (43 C) produced saffron with a safranal content up to 25 times more than that of saffron dried only at lower temperatures. Evidence was provided suggesting that drying with significant air flow reduced the safranal concentration. The results, moreover, indicated that high-temperature treatment had allowed greater retention of crocin pigments than in saffron dried at intermediate temperatures (46 C
58 C). The biochemical implications of the various treatments are discussed in relation to their potential for optimizing color and fragrance quality in the product. The effect of mild temperature on the main components responsible for saffron quality during dehydration was studied by Del Campo et al. (2010). Based on their results, crocetin esters were not as labile as the bibliography mentioned before. Saffron coloring capability increased from 40 C, without finding significant differences with 55 C. A similar behavior was obtained for picrocrocin, that was higher at the highest temperature but without significant differences with the immediate inferior conditions. However, at higher temperatures (e.g., 55 C) more volatile compounds, especially safranal, were generated during the dehydration procedure. The results of chromatographic analyses by Cossignani et al. (2014) also showed that samples dried in milder conditions had the lowest content of secondary metabolites such as crocins, picrocrocin, and safranal. Moreover, samples dried at 60 C for 55 minutes presented the highest contents of trans-crocin-4 and picrocrocin, while safranal was most prevalent compound in saffron dried at 55 C for 95 minutes. A detailed study by Tong et al. (2015) determined that the highest quality saffron is obtained when fresh saffron is treated at higher temperatures (no more than 70 C) for a long period by electric oven drying and vacuum oven drying. As mentioned before, microwave radiation as a mild drying method is an efficient way for improving the chemical profile of saffron in terms of safranal. The total safranal content increases when saffron is dehydrated at moderate temperatures using microwaves (Muzaffar et al., 2015). Microwave drying retains the maximum concentration of safranal as compared with samples dried under shade. Therefore, microwave drying could be the best drying method for saffron stigmas in order to retain its aroma. According to Maghsoodi et al. (2012), among saffron drying methods, microwave drying obtained the highest amount of safranal at 1000 W. Under these conditions the amounts of crocin and picrocrocin were also high. Results by Tong et al. (2015) also showed that the chemical contents in saffron treated by microwave drying were higher than the other methods used and that the time spent in the drying process was also less. However, the antioxidant activity of these samples was not stronger, which means that other chemical compounds were formed in the samples treated by electric oven drying and vacuum oven drying processes. The sarfanal content of the samples dried in a freeze dryer was found to be five times higher than the safranal content of the samples dried naturally under the sun. Crocin content of samples dried in a freeze dryer was about 40% higher than the crocin content of samples dried naturally under the sun. These results indicate that the safranal and crocin contents that define the quality and market value of commercial saffron were considerably higher in samples dried in a freeze dryer than in the samples dried traditionally under the sun (Acar et al., 2011).

17.10 Conclusion Drying must be done in a proper way to achieve saffron with proper color, aroma, and taste. A dehydration treatment changes the physical and biochemical properties of saffron spice, which is necessary to achieve desired attributes. The quantities of crocins, safranal, and picrocrocin in saffron depend mainly on the method used for drying the stigmas. In traditional methods using solar and air drying, the carotenoids in saffron can be degraded. However, the purplish red

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color and aroma in saffron are retained when the air drying method is used. In artificial drying methods, such as drying over charcoal, the organoleptic qualities and its purplish red color are preserved. Due to environmental concerns, a solar photovoltaic/thermal collector dryer has been developed in order to decrease the total drying time and energy consumption for saffron drying. IR heating is another drying method, which has some advantages such as lower drying time, higher energy efficiency, and lower environmental impact. The maximum values of crocin and safranal were obtained in saffron dried at the highest IR temperature. In the microwave drying method, heat conductivity is not a critical factor, and saffron can be dried in a shorter time and at a lower temperature. Therefore, saffron will have higher color strength, aroma, and taste, with better quality. The total safranal content increases when saffron is dehydrated using microwave drying. Freeze-dried saffron has the highest quality when compared with other, conventionally dehydrated products. Saffron processed in this manner contains high amounts of safranal and crocin and also has lower moisture content. However, the initial investment and operational costs are high, and the drying time is substantially long.

References Acar, B., Sadikoglu, H., Ozkaymak, M., 2011. Freeze drying of saffron (Crocus sativus L.). Dry. Technol. 14, 1622
1627. Acar, B., Sadikoglu, H., Doymaz, I., 2015. Freeze-drying kinetics and diffusion modeling of saffron (Crocus sativus L.). J. Food Process. Preserv. 39, 142
149. Aghbashlo, M., Mobli, H., Rafiee, S., Madadlou, A., 2013. A review on exergy analysis of drying processes and systems. Renew. Sustain. Energy Rev. 22, 1
22. Akhondi, E., Kazemi, A., Maghsoodi, V., 2011. Determination of a suitable thin layer drying curve model for saffron (Crocus sativus L) stigmas in an infrared dryer. Sci. Iran. 18, 1397
1401. Alonso, G.L., Varon, R., Salinas, M.R., Navarro, F., 1993. Auto-oxidation of crocetin and picrocrocin in saffron under different storage conditions. Boll. Chim. Farm. 132, 116
120. Alonso, G.L., Salinas, M.R., Garijo, J., 1998. Method to determine the authenticity of aroma of saffron (Crocus sativus L.). J. Food Prot. 61, 1525
1528. Carmona, M., Zalacain, A., Pardo, J.E., Alvarruiz, A., Alonso, G.L., 2005. Influence of different drying and aging conditions on saffron constituents. J. Agric. Food Chem. 53, 3974
3979. Carmona, M., Zalacain, A., Alonso, G.L., 2006. The Chemical Composition of Saffron: Color, Taste and Aroma. Editorial Bomarzo SL, Albacete, Spain. Carmona, M., Zalacain, A., Salinas, M.R., Alonso, G.L., 2007. A new approach of saffron aroma. Crit. Rev. Food Sci. Nutr. 47, 145
159. Celma, A.R., Rojas, S., Lopez-Rodrı´guez, F., 2008. Mathematical modeling of thin-layer infrared drying of wet olive husk. Chem. Eng. Prog. 47, 1810
1818. Cossignani, L., Urbani, E., Stella Simonetti, M., Maurizi, A., Chiesi, C., Blasi, F., 2014. Characterisation of secondary metabolites in saffron from central Italy (Cascia, Umbria). Food Chem. 143, 446
451. Del Campo, C.P., Carmona, M., Maggi, L., Kanakis, C.D., Anastasaki, E.G., Tarantilis, P.A., et al., 2010. Effects of mild temperature conditions during dehydration procedures on saffron quality parameters. J. Sci. Food Agric. 90, 719
725. Feng, H., Yin, Y., Tang, J., 2012. Microwave drying of food and agricultural materials: basics and heat and mass transfer modeling. Food Eng. Rev. 4, 89
106. Gregory, M.J., Menary, R.C., Davies, N.W., 2005. Effect of drying temperature and air flow on the production and retention of secondary metabolites in saffron. J. Agric. Food Chem. 53, 5969
5975. Iglesias, H.A., Chirife, J., 1982. Hand Book of Food Isotherms. Academic Press, New York, pp. 170
175. Maghsoodi, V., Kazemi, A., Akhondi, E., 2012. Effect of different drying methods on saffron (Crocus sativus L.) quality. Iran. J. Chem. Chem. Eng. 31, 85
89. Mortezapour, H., Ghobadian, B., Minaei, S., Khoshtaghaza, M.H., 2012. Saffron drying with a heat pump
assisted hybrid photovoltaic
thermal solar dryer. Dry. Technol. 30, 560
566. Mortezapour, H., Ghobadian, B., Khoshtaghaza, M.H., Minaei, S., 2014. Drying kinetics and quality characteristics of saffron dried with a heat pump assisted hybrid photovoltaic-thermal solar dryer. J. Agr. Sci. Tech. 16, 33
45. Muzaffar, S., Zaman Khan, K., Riaz, M., Mir, J.A., Ahmed, A., 2015. Aroma of Kashmir saffron (Crocus sativus L.) and variation of safranal content by different drying methods. J. Chem. Pharm. Res. 7, 111
115. Ordoudi, E., Tsimidou, M., 2004. Saffron quality: effect of agricultural practices, processing and storage. In: Dris, R., Jain, S.M. (Eds.), Production Practices and Quality Assessment of Food Crops. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 209
260. Rahman, M.D.S., 1995. Hand Book of Food Properties. CRC Press, New York, pp. 2
11. Raina, B.L., Agarwal, S.G., Bhatia, A.K., Gaur, G.S., 1996. Changes in pigments and volatiles of saffron (Crocus sativus L.) during processing and storage. J. Sci. Food Agric. 71, 27
32. Raines Ward, D., 1988. Flowers are a mine for a spice more precious than gold. Smithsonian 19, 105
110. Sadikoglu, H., Liapis, A.I., 1997. Mathematical modelling of the primary and secondary drying stages of bulk solution freeze-drying in trays: parameter estimation and model discrimination by comparison of theoretical results with experimental data. Dry. Technol. 15, 791
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Sadikoglu, H., Liapis, A.I., Crosser, O.K., 1998. Optimal control of the primary and secondary drying stage of bulk solution freeze drying in trays. Dry. Technol. 16, 399
431. Sadikoglu, H., Ozdemir, M., Seker, M., 2003. Optimal control of the primary drying stage of freeze drying process in vials using variational calculus. Dry. Technol. 21, 1307
1331. Sadikoglu, H., Ozdemir, M., Seker, M., 2006. Freeze-drying of pharmaceutical products: research and development needs. Dry. Technol. 24, 849
861. Schubert, H., Regie, M., 2006. Novel and traditional microwave applications in the food industry. In: Willert-Porada, M. (Ed.), Advances in Microwave and Radio Frequency Processing. Springer, New York, pp. 259
270. Tammaro, F., 1999. Saffron (Crocus sativus L.) in Italy. In: Negbi, M. (Ed.), Saffron Crocus sativus L. Medicinal and Aromatic Plants Industrial Profiles. Harwood Academic Publishers, Amsterdam, pp. 53
61. Tong, Y., Zhu, X., Yan, Y., Liu, R., Gong, F., Zhang, L., et al., 2015. The influence of different drying methods on constituents and antioxidant activity of saffron from China. Int. J. Anal. Chem. 2015, 1
8. Torki-Harchegani, M., Ghanbarian, D., Maghsoodi, V., Moheb, A., 2017. Infrared thin layer drying of saffron (Crocus sativus L.) stigmas: mass transfer parameters and quality assessment. Chinese J. Chem. Eng. 25, 426
432. Tsimidou, M., Biliaderis, C.G., 1997. Kinetic studies of saffron (Crocus sativus L.) quality deterioration. J. Agric. Food Chem. 45, 2890
2898. Zanzucchi, C., 1987. La ricerca condotta dal consorzio comunale Parmaensi sullo zafferano (Crocus sativus L.). In: A. Bezzi (Ed.), Atti Convegno sulla coltivazione delle piante officinali, 9
10 ottobre, Istituto Sperimentale per l’Assestamento Forestale e per l’Alpicoltura, Villazzano (Trento), pp. 347
395 (in Italian). Zarein, M., Samadi, S.H., Ghobadian, B., 2015. Investigation of microwave dryer effect on energy efficiency during drying of apple slices. J. Saudi Soc. Agric. Sci. 14, 41
47. Ziaforoughi, A., Yousefi, A.R., Razavi, S.M.A., 2016. A comparative modeling study of quince infrared drying and evaluation of quality parameters. Int. J. Food Eng. 12, 1
9.

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Chapter 18

Saffron packaging Arash Koocheki Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Chapter Outline 18.1 18.2 18.3 18.4 18.5 18.6

Introduction Saffron packaging Paper and paperboard Aluminum foil Glass Low-density polyethylene

18.1

301 302 302 302 302 303

18.7 High-density polyethylene 18.8 Modified atmosphere packaging 18.9 Nanosilver composite antimicrobial packaging 18.10 Edible and biodegradable films 18.11 Conclusion References

303 303 304 304 304 305

Introduction

Food packaging is one of the most important processes, which preserve the quality of food products during storage, transportation, and end use (Kelsey, 1985). The quality of food might be deteriorated biologically, chemically, and physically, when the product is distributed. Therefore food packaging is needed to extend the shelf-life and maintain the quality and safety of the food products (Han, 2014). The packaging mainly protects the food from environmental physical damage, humidity, oxygen, light, and, to some extent, temperature. Packaging can also protect foods against microbial contamination. Good package integrity is also required to protect food against loss of hermetic condition and microbial penetration (Yam and Lee, 2012). Although food products manufactured by retorting or aseptic processing do not require refrigeration shelf-stable packaging is needed as a barrier against the invasion of microorganisms (Han, 2014). Marketing is another important function of food packaging, which provides traceability, indications of tampering, and portion control (Marsh and Bugusu, 2007). Packaging is the main communication element for the product at the point-of-sale and allows the consumer to appreciate the position chosen by the brand (Pantin-Sohier, 2009). Therefore all food packaging must communicate with the consumer. Not only must the contents be identified, but the packaging should contribute to sales and marketing efforts. Packaging design is one of the important tools in modern marketing and consumer acceptability of the product. The color of the package has a psychological effect on the behavior of buyers in different ways (Gilaninia et al., 2013). According to Dourandish et al. (2017) the information on the label of package regarding the internal and international standards and also about the saffron nutritional values have the greatest impact on the consumer acceptability. The brand labeled on saffron package is another important issue from the perspective of the consumers. Therefore producers and suppliers of saffron should pay more attention to the features and information on the package design. The attributes of food and packaging materials have a direct influence on the quality of the packaged food. Most food products deteriorate in quality due to mass transfer phenomena, such as moisture absorption, oxygen invasion, flavor loss, undesirable odor absorption, and the migration of packaging components into the food (Debeaufort et al., 1998; Kester and Fennema, 1986). Therefore in order to find a proper package, a number of considerations must be taken into account. First, the package must provide the optimum protective properties to keep the product in good condition for its anticipated shelf-life. Second, the package should have good shape and size and its graphics must attract the eyes of the purchaser (Paine and Paine, 1992). Although preservation, convenience, and other basic functions of packaging are certainly important, its disposal should also be considered an important aspect of packaging development (Han, 2014). Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00018-6 © 2020 Elsevier Inc. All rights reserved.

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For herbs and spices it is important to know how to store them effectively in order to reduce their deterioration during storage. Most herbs are marketed in dried form in order to avoid product deterioration over time. According to King (2006) several factors such as ease of opening, reseal features, pouring qualities, protection from light, transparency, tamper-proof construction, and physical characteristics of outside surface including appearance must be taken into account when choosing a suitable packaging material for herbs and spices.

18.2

Saffron packaging

After drying and sorting the stigmas, saffron is ready for packaging and release. The most important factors decreasing the quality of stigmas during storage are climatic influences that cause physical and chemical changes [ultraviolet (UV) light, moisture vapor, oxygen, and temperature changes]; contamination (by microorganisms, insects, or soils); and also saffron packaging materials. These factors should be considered and most of them should be avoided during packaging of saffron stigmas. The primary purpose of stigmas packaging is to preserve the flavor and color and also to keep the product in good condition until it reaches consumers (King, 2006). In order to reduce oxidation reactions it is important to avoid high-temperature storage, utilize packaging with low oxygen permeability and use modified atmosphere packaging. Most common packages used for these purposes are glassware, polyethylene bags, polyethylene jars, and aluminum-layered bags. In most cases saffron is placed inside cardboard, wood, or metal boxes to prevent the package from pressing and breaking during shipping.

18.3

Paper and paperboard

Paper and paperboard are felted sheets, usually composed of plant fibers or other fibrous materials which are used to make package (Riley, 2012). They are categorized by the weight or thickness of the product, with paper being lighter than paperboard (Kim et al., 2014). These are the least expensive packages for whole spices. Paper and paperboard packages are easy collected, reused, and recycled. The excellent stiffness and deadfold of papers and paperboards allow them to be used for bags, ensuring their creases are sharp and they stand erect on shelves. The tensile strength of paper and paperboard is high and their extensibility low, allowing for good constant tension to be applied when printing and laminating papers and during the manufacture of corrugated board. This high printability is ideal for displaying product information and nutritional value for marketing purposes (Kim et al., 2014). One of the negative properties of paper and paperboard is that they have high permeability to flavor components and gases (King, 2006) and absorb moisture vapor and water (Riley, 2012). Therefore paper and paperboard are unsuitable for ground spices and saffron. To improve the gas or wet barrier properties and the strength of paper and paperboard, they can easily be combined with other materials such as oil, wax, polymers (plastics), and metals through coating or lamination (Kim et al., 2014). Wax coating on the outside of the packaging improves attractiveness and resistance to water; while polyethylene coating inside gives extra protection and sealability to papers and paperboards (King, 2006). Paper and paperboard are mainly used as secondary packages for saffron packaging and thus are not directly in contact with the product inside the package. Therefore there is no need to combine these papers with other materials or use coatings or lamination with plastics.

18.4

Aluminum foil

Metal can provide good protection against physical damage and provide a good barrier against water, oxygen, and gases. Among metals aluminum has a number of important characteristics such as its light weight, high yield strengths in its alloy form, and resistance to corrosion; additionally, it is easy to form and is a good conductor of heat and electricity (Kerry, 2012). When exposed to air, aluminum forms a transparent oxide layer, which prevents further oxidation. As well as being resistant to corrosion, aluminum is nonabsorbent and thus an effective barrier against gases and liquid. Its resistance to gas transmission is essential to protect the delicate flavor of many spices. Aluminum does not generate toxic residues or react with most chemicals including the majority of foods. It is not transparent and is ideal for spices that need protection from light (King, 2006). Therefore aluminum foils have great potential for packaging saffron. Aluminum can easily be recycled and used again for packaging, which is another important advantage of this metal (Kerry, 2012).

18.5

Glass

Glass is one of the oldest packaging materials and is made of silica (quartz), which is the principal component of sand (Grayhurst, 2012). Glass surfaces may be treated with titanium, aluminum, or zirconium compounds to increase their

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strength and enable thinner and lighter containers (King, 2006). Using boron compounds (borax, boric oxide) give high heat-shock resistance to the glass. The majority of glass used for food containers can be easily reused and recycled (Han, 2014). As a food packaging material, glass has both disadvantages and advantages. One of the advantages of glass is that it does not react with food to produce hazardous or undesirable compounds. Glass has also good barrier properties against gases and chemicals and can tolerate high temperatures. The transparency of glass is preferred by consumers because it provides visibility of the saffron inside. However, glass is prone to breakage upon physical impact and high pressure. Another disadvantage is that translucent materials such as glass have little protection against photooxidation and the light energy can pass through the material and result in photochemical reactions, which lead to product discoloration. Amber glass is able to filter out UV light and is primarily used for UV-sensitive products.

18.6

Low-density polyethylene

Low-density polyethylene (LDPE) is one of the most widely used packaging plastics. It is translucent in appearance, heat sealable, chemically inert, odor free, soft, and flexible with good elongation before breakage and good puncture resistance (Emblem, 2012). LDPE has a fair moisture barrier property but has relatively high gas permeability (King, 2006). It is also characterized by its fair machinability, good oil resistance, fair chemical resistance, good heat sealing characteristics, and low cost. Since LDPE softens at around 100 C, it is an economical polymer to be processed and is readily heat sealable. Linear low-density polyethylene (LLDPE) is produced as either a homopolymer or copolymer having comonomer alkenes such as butane, hexane, and octane (Kim et al., 2014). It has similar properties to LDPE, although it is tougher with slightly better barrier properties. Due to the increased regularity of the structure and narrower molecular weight distribution, the mechanical properties of LLDPE are improved compared to LDPE at the same density. When compared with LDPE, LLDPE requires slightly more energy to be heat sealed and its operating range for sealing is narrower. Therefore controlling the seal temperature on the packaging machine is more critical. LLDPE is also a little more transparent than LDPE (Emblem, 2012). Rains et al. (1996) packed dried saffron in 10-gauge polyethylene bags and evaluated its coloring power during storage at ambient temperature. The loss of crocin at this condition varied based on the initial moisture content of the stigmas. Samples with initial moisture content of 5% deteriorated more slowly during storage in polyethylene bags. According to Karazhiyan et al. (2012) the moisture, crocin, picrocrocin, and microbial content decreased during storage of saffron in polyethylene bags for 1 year. The largest variations were observed until 8 months of storage and after that the variations were not significant. LDPE pouches could also be used for storage of saffron flowers (Shoormij et al., 2012). LDPE pouches maintained the physical and quality properties of saffron flowers especially when stored at 0 C. Similar to these findings results by Salari et al. (2010) also showed that the microbiology of saffron packaged in polyethylene polymers decreased during 1 year of storage. They reported that this decrease in microbial content was due to the increase in the content of safranal produced from picrocrocin.

18.7

High-density polyethylene

High-density polyethylene (HDPE) is a translucent polymerized film that has higher crystallinity and provides a good barrier against gas and water compared to LDPE. It is stronger, thicker, less flexible, and more brittle than LDPE (King, 2006). It is waterproof and has excellent resistance to a wide range of chemical compounds. The melting temperature (Tm) of this film is around 135 C and thus it withstands boiling water. Therefore HDPE is suitable as a film for boil-in-the-bag foods (Emblem, 2012). However, it is prone to environmental stress cracking.

18.8

Modified atmosphere packaging

Modified atmosphere packaging (MAP) is a term that implies the addition or removal of gases from packages in order to manipulate the levels of gases such as oxygen, carbon dioxide, nitrogen, ethylene, etc. (King, 2006). MAP is mainly used to extend the shelf-life of food products and to prevent any undesirable changes in foods. MAP dramatically extends the shelf-life of packaged food products, and in some cases MAP products do not require any further treatment or any special care during distribution. The use of MAP for storage of Spanish saffron showed a marked decrease in its color loss during storage (Khodabakhsh, 2010). Results by Jouki and Khazaei (2013) also showed that during storage of Iranian saffron under

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modified atmosphere, the rate of decrease in the content of microorganisms was higher. Therefore in situations where chemical additives may be of concern, MAP can be used effectively for packaging saffron.

18.9

Nanosilver composite antimicrobial packaging

Nanotechnology has been touted as the next revolution in many industries, including food processing and packaging. Addition of nanocomposites or nanoparticles (e.g., silver, titanium dioxide, silicon dioxide, nanoclay) into food packaging materials is done to ensure better protection of foods by increasing barrier properties, blocking UV light, improving mechanical and heat-resistance properties, and developing antimicrobial and antifungal surfaces (Chau et al., 2007). Antimicrobial packaging is used to extend the shelf-life of food products through prevention of spoilage and pathogenic microorganism growth. Antimicrobial packaging systems are able to kill or inhibit microorganisms that cause food spoilage or foodborne illnesses (Han, 2003). Sliver and porous materials complexes with silver such as zeolite used as antimicrobial particles besides polymeric films or surface coating (Pehlivan et al., 2005). However, nanosilver particles have shown notable antimicrobial activity and have higher Ag 1 ions release rate compare to that of bulk silver (Echegoyen and Nerin, 2013; Lee et al., 2012; Reidy et al., 2013). Polyethylene
nanosilver composites packaging can prevent or limit the microbial putrefaction of saffron (Eslami et al., 2016). However, since the release rate is mainly under the control of several factors such as moisture, there is a delay in the antibacterial effects of nanosilver due to the dry nature of saffron. Therefore although packaging with polymer
nanosilver can maintain growth inhibition or killing of microorganisms in dry products, these effects are not similar to those recognized for liquid and semiliquid foods, like beverages. Among the microorganisms, Salmonella enteritidis and enterococci are more susceptible to silver, while fungi are less sensitive to the agent. Nanosilver particle film packaging could also increase the flavor and aroma of saffron during storage (Hamid Sales et al., 2012). For more details see Chapter 19, Assessment and monitoring of saffron microbiological criteria.

18.10 Edible and biodegradable films Synthetic polymer materials used in the packaging industries are nonbiodegradable and therefore lead to environmental pollution, posing serious ecological problems. Biopolymer films have received increased attention because of their biodegradability (Monjazeb-Marvdashti et al., 2017). Antimicrobial agents can also be added to these polymers for controlling the contamination of food packed inside (Monjazeb-Marvdashti et al., 2019). Edible and biodegradable natural polymer films offer alternative packaging with lower environmental cost. Biodegradable films are generally made from biopolymers such as polysaccharides, proteins, and lipids (Seyedi et al., 2014). These materials have been widely considered as prospective replacers of synthetic polymers such as plastics. However, these biopolymers are not plastic, and it is not feasible to produce biopolymers using existing machinery in plastic packaging plants. These environmentally friendly films must meet a number of specific functional requirements such as moisture barrier, solute and/or gas barrier, color and appearance, mechanical and rheological features, and nontoxicity (Guilbert, 2000). Generally, biopolymeric materials provide high oxygen and flavor barrier properties but lower water barrier properties, mostly depending on moisture content and the amount of plasticizers (Kim et al., 2007). The use of biopolymers as a packaging material is limited due to their production costs, functionality, and compatibility with other polymers in current recycling streams (Roberts et al., 2011). Films made from only one kind of natural polymer display good properties in some aspects, while poor in others (Seyedi et al., 2015). In order to improve the film properties simultaneously, two or more biopolymers can be combined. Functional properties of composite films depend on their composition and film-forming procedure (Oses et al., 2009). These blends can strongly improve the physical and chemical properties of films prepared by each polymer (Ebrahimi et al., 2016). Films prepared from binary polymeric blends have different structures, which influence their final properties (Guerrero et al, 2013). Fatty acids, lipids, and waxes are commonly used in polysaccharide and protein edible films to reduce their water-vapor permeability since these materials are hydrophobic and thus are good barriers against moisture migration (Seyedi et al., 2015).

18.11 Conclusion Barrier properties including permeability of gases, water vapor, aromas, and light are vital factors for maintaining the quality of saffron. Most common packages used for saffron are glassware, LDPE, HDPE, and aluminum-layered bags. Among these packages, LDPE is the most common while glass container does not react with saffron and has good

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barrier properties against gases. In order to increase the shelf-life of saffron, MAP could be used, which reduces the color loss during storage. To ensure better protection of saffron, nanosilver particles could be added to the package. Polymer
nanosilver can inhibit growth of or kill microorganisms. The substitution of artificial chemical ingredients in packaging materials with natural ingredients is always attractive to consumers. Biopolymer films are a great alternative to synthetic polymer materials due to their biodegradability. Therefore combinations of biopolymers with good barrier properties could be used for saffron packaging in the future.

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409. Guilbert, S., 2000. Edible films and coating and biodegradable packaging. Bull. Int. Dairy Fed. 346, 10
16. Hamid Sales, E., Motamedi Sedeh, F., Rajabifar, S., 2012. Effects of gamma irradiation and silver nano particles on microbiological characteristics of saffron, using hurdle technology. Ind. J. Microbiol. 52, 66
69. Han, J.H., 2003. Antimicrobial packaging materials and films. In: Ahvenainen, R. (Ed.), Novel Food Packaging Techniques. Woodhead Publishing, Cambridge, UK. Han, J.H., 2014. A review of food packaging technologies and innovations. In: Han, J.H. (Ed.), Innovations in Food Packaging, second ed. Academic Press, Waltham, MA. Jouki, M., Khazaei, N., 2013. Effects of low-dose γ-irradiation and modified atmosphere packaging on shelf-life and quality characteristics of saffron (Crocus sativus L.) in Iran. Food Sci. Biotechnol. 22 (3), 687
690. Karazhiyan, H., Salari, R., Vazirzadeh, B., 2012. Effect of storage time on physiochemical and microbial properties of saffron. J. Agric. Food. Tech. 2 (4), 61
68. Kelsey, R.J., 1985. Packaging in Today’s Society, third ed Technomic, Lancaster, PA. Kerry, J., 2012. Aluminium foil packaging. In: Emblem, A., Emblem, H. (Eds.), Packaging Technology: Fundamentals, Materials and Processes. Woodhead Publishing, Cambridge, UK. Kester, J.J., Fennema, O.R., 1986. Edible films and coatings: a review. Food Technol. 48 (12), 47
59. Khodabakhsh, M.M., 2010. Effect of Modified Atmosphere Packaging and Antioxidants on the Color Stability of Saffron (Crocus sativus L.) Under Various Temperature Conditions (Thesis), UMI Number, EP30770, Chapman University, Orange, CA. Kim, Y.T., Hong, Y.S., Kimmel, R.M., Rho, J.H., Lee, C.H., 2007. New approach for characterization of gelatin biopolymer films using proton behavior determined by low field 1H NMR spectrometry. J. Agric. Food Chem. 55, 10678
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Oses, J., Fabregat-Vazquez, M., Pedroza-Islas, R., Tomas, S.A., Cruz-Orea, A., Mate, J.I., 2009. Development and characterization of composite edible films based on whey protein isolate and mesquite gum. J. Food Eng. 92, 56
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71. Pehlivan, H., Balkosee, D., Ulku, S., Tihminlioglu, F., 2005. Characterization of pure and silver exchanged natural zeolite filled polypropylene composite films. Compos. Sci. Tech. 65, 2049
2058. Rains, B.L., Agarwal, S.G., Bhatia, A.K., Gaur, G.S., 1996. Changes in pigments and volatiles of saffron (Crocus sativus L.) during processing and storage. J. Sci. Food. Agric. 71, 27
32. Reidy, B., Haase, A., Luch, A., Dawson, K.A., Lynch, I., 2013. Mechanisms of silver nanoparticle release, transformation and toxicity: a critical review of current knowledge and recommendations for future studies and applications. Materials 6, 2295
2350. Riley, A., 2012. Paper and paperboard packaging. In: Emblem, A., Emblem, H. (Eds.), Packaging Technology: Fundamentals, Materials and Processes. Woodhead Publishing, Cambridge, UK. Roberts, D., Gangemi, J.D., Kim, Y.T., 2011. Articles of manufacture from renewable resources. United States patent application US201100847A1. Salari, R., Habibi Najafi, M.B., Karazhiyan, H., Vazirzadeh, B., 2010. Evaluation of physiochemical and microbial changes in saffron during one year storage. J. Food Sci. Technol. 2, 35
43. Seyedi, S., Koocheki, A., Mohebbi, M., Zahedi, Y., 2014. Lepidium perfoliatum seed gum: a new source of carbohydrate to make a biodegradable film. Carbohyd. Polym. 101, 349
358. Seyedi, S., Koocheki, A., Mohebbi, M., Zahedi, Y., 2015. Improving Lepiduim perfoliatum seed gum biodegradable film barrier properties using stearic or palmitic acids. Int. J. Biol. Macromol. 77, 151
158. Shoormij, M., Einafshar, S., Niazmand, R., Sharayei, P., 2012. The effect of storage temperature and packaging material on the quantity, quality and microbial properties of modified atmosphere packaging of saffron flower. J. Res. Innov. Food Sci. Technol. 1, 283
292. Yam, K.L., Lee, D.S., 2012. Emerging food packaging technologies: an overview. In: Yam, K.L., Lee, D.S. (Eds.), Emerging Food Packaging Technologies: Principles and Practice. Woodhead Publishing, Cambridge, UK.

Chapter 19

Assessment and monitoring of saffron microbiological criteria Elnaz Milani Food Science and Technology Research Institute, ACECR, Mashhad, Iran

Chapter Outline 19.1 Introduction 19.2 Microbial critical point in saffron 19.2.1 Microbiological analysis 19.3 Monitoring harvesting of saffron flowers 19.4 Monitoring transportation of saffron flowers 19.5 Microbial decontamination of saffron by different postharvest processes 19.5.1 Effect of drying processes on microbiological quality of dried stigma 19.5.2 Effect of cold plasma process on microbiological quality of dried stigma 19.5.3 Effect of ozone treatment on microbiological quality of dried stigma

19.1

307 308 308 309 310 310 311 313

19.5.4 Effect of irradiation treatment on microbiological quality of dried stigma 19.5.5 Infrared irradiation treatment 19.6 Saffron packaging 19.6.1 Modified atmosphere packing 19.7 Effect of antibacterial packaging on microbiological quality of dried stigma 19.8 Effect of Hurdle technology on microbiological quality of dried stigma 19.9 Conclusion References Further reading

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Introduction

Saffron is globally the most expensive agricultural product, and it has been used intensively for flavoring and coloring foods, for dyeing textiles, and for medical purposes since ancient times (Abdullaev, 2002). It has been estimated that over 900,000 flowers and one hectare of land are needed to produce 6 kg of saffron (Souret and Weathers, 2000). At global scale, saffron flower has a production of 867,659 tons (FAOSTAT, 2016). Contamination of saffron is defined as “the undesired introduction of impurities of a chemical or microbiological nature, or of foreign matter, into starting material, intermediate product or finished product during production, sampling, packaging or repackaging, storage or transport.” Concerns have been raised repeatedly regarding the contamination of saffron. Harvesting process of saffron is considered as a critical control point with major effects on quality and safety parameters. Inappropriate postharvest processes will result in reduced quality. Considering that saffron corms require handplanting, hand-picking, and hand-separating, it is clear that saffron production is an extremely time-consuming and labor-intensive cultivation process (Asimopoulos et al., 2013; Gresta et al., 2008; Winterhalter and Straubinger, 2000). Thus, this crop is prone to microbial contamination during collection and processing as well as from dust and wastewater (Sales et al., 2012). The aim of this chapter is to provide a summary and critical evaluation of saffron contamination during the different stages of production.

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00019-8 © 2020 Elsevier Inc. All rights reserved.

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Microbial critical point in saffron

Several microorganisms detected in saffron have the potential to cause human illness, including aflatoxin-producing fungi (e.g., Aspergillus spp.), Bacillus cereus, Clostridium perfringens, Escherichia coli, Salmonella spp., and Staphylococcus aureus (Cho et al., 2008; Cosano et al., 2009). The ability of salmonella to survive drying treatments and subsequent storage in low-water-activity food ingredients increases the risk of infection especially when added to ready-to-eat foods (Ulbricht et al., 2011). Outbreaks of salmonellosis associated with the consumption of contaminated spices or herbs have been reported (Vibha et al., 2006). Low levels of contamination by B. cereus and C. perfringens in saffron are frequent because of the natural habitat in the soil environment. Both of these bacterial species cause foodborne disease when ingested in large numbers, which may result from growth in food matrices, usually as a result of poor temperature and time control (Aguilera et al., 2005; Banerjee and Sarkar, 2003). S. aureus also causes food poisoning and is most likely to originate from contact with food handlers during harvesting, processing, and storage, and its absence reflects good hygiene practices (Jung et al., 2008). E. coli is a fecal contamination indicator, and it is frequently found in soil and water contaminated with human sewage or animal slurry, but can also originate from wildlife fecal contamination (Leifert et al., 2008). C. perfringens is a spore-forming anaerobic microorganism that may cause foodborne disease by the action of enterotoxin CPE. The bacterium grows in anaerobic conditions. The disease is characterized mainly by watery diarrhea with an incubation time of 8
24 hours and duration of the disease symptoms is 24 hours at most. The disease is selflimiting (Aguilera et al., 2005).

19.2.1 Microbiological analysis 19.2.1.1 Classical culturing method Microbiological studies of saffron spice were performed according to the methods described by Cosano et al. (2009) and Khazaei et al. (2011). For this purpose, saffron stigma was chosen randomly from collected samples of saffron flowers. Then 2 g of saffron was dissolved in 1 L of water, after which the stigma were removed from the liquid by passing it from a filter and then the obtained liquid was used. Test samples were agitated and 1 mL of test sample was diluted in sterile ringer solution (9 mL) to make primary dilution of 1021. A series of dilution up to countable range were then prepared; 1 mL of each dilution was added into the sterile plate. Plating was always done in duplicate, and the mean of countable colonies was calculated. For detection of total count of aerobic mesophilic bacterial, diluted samples were inoculated in plate count agar media and incubated for 48 hours at 37 C. The number of viable bacteria colonies is expressed as microbial counts (log CFU g21). S. aureus was detected using 0.1 mL of each dilution in plate containing Baird
Parker agar and incubated for 48 hours at 37 C (Banerjee and Sarkar, 2003). Enumeration of yeasts and molds was done by pour plates with 0.1 mL of each dilution in plates containing potato dextrose agar, incubation at 28 C for 7
10 days (Abou Donia, 2008; Cosano et al., 2009; Khazaei et al., 2011). Salmonella screening was carried out while 1 mL aliquot was inoculated in preenrichment culture of peptone water and selenite and rappaport vassiliadis broth for 24 hours at 37 C and 45 C, respectively (Milani et al., 2016). To investigate the numerate of enterobacteriaceae, coliforms, and E. coli, an aliquot of dilutions were inoculated in violet red bile glucose agar, violet red bile agar with lactose, Coli-ID agar, and incubated at 30 C, 37 C, and 45 C, respectively. The maximum possibility number of coliform was determined by the three tubes in lactose broth media for 24 hours at 37 C and 1 mL of each tube, which was positive for gas production, was added to another tube containing brilliant green broth (Cosano et al., 2009). B. cereus were enumerated selectively by plate count in mannitol egg yolk polymyxin agar for 48 hours at 30 C (Milani et al., 2016). The presence of sulfite-reducing sporulated bacteria and C. perfringens, determined using iron sulfite agar plates, were 1 mL of each dilution incubated for 72 hours at 37 C and 45 C in anaerobic conditions, respectively (Milani et al., 2016). The microbiological profile of saffron spice was investigated by Cosano et al. (2009). In their study, 79 samples were obtained from the main producer countries, namely Iran, Italy, Greece, Spain, and Morocco. An objective of this study was to set new criteria for the microbiological quality of saffron. Salmonella was not detected in any sample, and E. coli was only found in five samples. Enterobacteriaceae were frequently found. Aerobic sporulated bacteria were also common, but only three samples contained B. cereus at low levels (200 CFU g21). C. perfringens counts

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were also very low, with only one sample reaching 100 CFU g21, an acceptable value. Overall, microbial contamination in saffron was markedly lower than it was in other spices.

19.2.1.2 Rapid assessment by molecular analysis The traditional methods are time consuming, labor intensive, costly, and require mycological expertise. Therefore, it is necessary to develop methodologies that are relatively rapid, highly specific, and feasible alternatives to existing methods. Polymerase chain reaction (PCR) is used to detect possible nonviable contaminated microflora. Detection of salmonella and E. coli in saffron samples based on PCR was investigated by Cosano et al. (2009). It was used after the preenrichment culture process as described earlier. PCR amplifications were carried out with TaqMan polymerase using these primers: 59-CGGTGGTTTTAAGCGTACTCTT-39 and 259-CGAATATGCTCCACAAGGTTA-39 for amplification of the invA gene of salmonella (Fratamico, 2003) and 59-AAAACGGCAAGAAAAAGCAG-39 and 59ACGCGTGGTTACAGTCTTGCG-39 for amplification of the uidA gene of E. coli (Bej et al., 1991). Products of amplification were analyzed by agarose gel electrophoresis. Salmonella Typhimurium LT2 and E. coli ATCC 29213 were used as controls, and amplification products compatible with the expected sizes of 796 and 1476 bp, respectively, were obtained. Aflatoxins are secondary metabolites primarily produced by two fungal species, Aspergillus flavus and Aspergillus parasiticus. Rapid detection of aflatoxigenic fungi in saffron using PCR was carried out by Noorbakhsh et al. (2009). Samples were assayed in order to isolate aflatoxin-producing fungi. Template DNA was extracted from the fungal mycelia and harvested from freshly growing cultures in potato dextrose broth. The PCR was used to amplify a part of the aflatoxin regulatory gene, aflR1, from aflatoxigenic fungal genomic DNA. The sequence of primers were 5AACCGCATCCACAATCTCAT-3 and 5-AGTGCAG TTCGCTCAGAACA-3, respectively. The majority of isolated fungi were saprophytes, which normally originate from soil during harvesting and postharvesting.

19.3

Monitoring harvesting of saffron flowers

Picking flowers takes place in the fall, lasts only 2
3 weeks, and picking is required almost daily. The flowers are picked by hand rapidly in the morning before the heat of the day, while the blossoms are thrown away. Once this has happened they cannot readily be separated into their constituent parts. Saffron is also very hygroscopic, with exposure to moisture creating a risk of spoilage of the product. Alternatively, during flower harvest, the whole flowers are dried and stigmas are picked by hand from the dried flowers later (Gresta et al., 2008). Since saffron flowers are usually harvested by hand, there is a high risk of contamination of the product with different microorganisms including bacteria and fungi. Microorganisms, especially spore-forming species, living in the soil and water resources are the other possible contaminants (Hamid-Sales et al., 2012). Today the technology of mechanical harvesting is focused on harvesting plants of large crops (e.g., wheat, corn) using special mowers large in size and high in cost. But the harvesting of vegetables, fruits, and other crops (e.g., saffron) depends primarily on human labor, which affacts the cost of production, the product’s quality, as well as the safety of workers picking crops that have been sprayed with pesticides (e.g., greenhouses). The main reasons for the shortage of automated solutions are the difficulty in tracking the crops and the difficulty in simultaneously cutting and collecting the crops without damaging them. The study about mechanical way of stigmata harvesting is limited (Asimopoulos et al., 2013). Mechanical cutting is possible theoretically but difficult in practice (Ruggiu and Bertetto, 2006). One common method is to pick up the stigmas from the flower or with a fingernail. One stigma of saffron weighs about 2 mg, each flower has three stigmas, and 150,000 flowers are required to produce 1 kg spice. Harvesting the flowers and separation of stigmas from the flower is the most difficult stage or production. It is time consuming, laborious, and makes saffron an expensive spice. Picking of 1000 flowers requires 45
55 minutes, and another 100
130 minutes is required to remove the stigmas for drying. Thus, 370
470 hours is required to produce 1 kg of dried saffron. Automation of harvesting and postharvesting of saffron flowers increases harvest efficiency and consequently reduces the final cost of the product. Dimitriadis and Brighton (2010) designed a machine that operates by separating the petals, stigmas, and stamens from the plant individually in the field using a combination of pneumatic and mechanical processes. Ruggiu and Bertetto (2006) developed a prototype for harvesting saffron. Javari and Bakhshipour (2010) developed a suitable algorithm for recognizing and locating saffron flowers using machine vision. Gracia et al. (2009) presented a new machine for automated cutting of saffron flowers in order to obtain their stigmas. Emadi and Yarlagadda (2008)

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designed a vertical wind tunnel for separation of stigmas from the other parts of saffron flowers using aerodynamic properties. Asimopoulos et al. (2013) developed a machine that can collect saffron flowers without damaging them. The design is based on assumptions regarding the location of the saffron plants, the dimensions, and the sensitivity due to the delicate nature of the flower. The vehicle is designed to operate at a speed comparable to that of a human and it can run approximately 4 hours without recharging. It is based on commonly available electrical and mechanical parts and communication and control devices. For more details refer to Chapter 11, Mechanization of saffron production, the chapter dedicated to mechanization of saffron production.

19.4

Monitoring transportation of saffron flowers

After harvesting, flower residues can be removed by hand in order to clean the product from dirt. Afterwards, saffron is kept in containers that protect its properties. The saffron flowers should be collected in sterile bags and not subjected to any washing or selection procedure. All the samples should be transported immediately under refrigerated conditions. Poor saffron flower storage conditions increase microbial contamination and decrease quality. Sharayee (2004) investigated the effects of harvesting and processing methods on microbial contamination and the qualitative characteristics of saffron. In this study, saffron flowers were harvested in three stages (bloom, semibloom, and full bloom) in zodiacal light. The stages of harvesting to drying were carried out in two levels: complete hygiene and traditional methods. The chemical and physical characteristics of the stigmas were measured before drying. All the treatments were dried in shadow (traditional), Spanish method, and oven method. After drying the samples were packaged in polyethylene (4.5 g) and stored at 10 C for 0, 6, and 12 months after drying. The results showed that the highest quality of saffron and the lowest level of microbial contamination were related to harvesting of saffron flowers buds form, the handling by clean containers, storage in the cold place away from sunlight and drying by the ordinary oven and the Spanish heater. Einafshar et al. (2014) studied a novel strategy to prolong the shelf-life of saffron flowers in packaging and subsequently exporting the fresh saffron flowers as cut flowers. Saffron flowers were packed in and stored in polyamidepolyethylene bags) and three gas combinations of CO2, O2, and N2 (30%, 5%, and 65%; 50%, 5%, and 45%; 70%, 5%, and 25%, respectively). Samples and control (without packaging) were kept at 0 C for 15 days. Every 3 days the concentrations of oxygen and carbon dioxide inside the packages, the yield of the product, physical (weight loss, percent of the flowers wilting, the length and diameter of fresh stigmas) and chemical properties (percent of moisture and the amount of crocin, safranal, and picrocrocin of dried stigma) were determined. The existence of E. coli and number of yeasts and molds were also monitored. The results showed that modified atmosphere packaging increased the shelf-life of saffron flowers. The chemical and physical qualities of control were lost strongly during 9 days. There were no evidence of E. coli growth in the samples. Enumeration of yeasts and molds showed that most treatments were seen the national standard of Iran (103 CFU g21). Azarpaahooh and Sharayei (2015) investigated the effect of cold storage on saffron quality. In the study, saffron flowers were harvested at semibloom stage and placed in plastic baskets. The flowers were stored at 0 C, 4 C, 8 C, and 21 C for 2, 4, 7, 14, and 21 days, respectively. During storage, weight loss percentage was calculated. In addition, the stigmas were separated from the styles and dried in an oven at 60 C. The results indicated that weight loss percentage increased with increasing storage thickness, temperature, and storage duration; however, bioactive compounds reduced. These values at 8 C and 21 C were not in the standard range; therefore these temperatures were not found suitable for flower storage. Although coliform contamination increased during storage days, the value was acceptable after 7 days. According to the results, it is recommended to store saffron flowers in baskets with 10 cm thickness accumulation at 0 C for 7 days.

19.5

Microbial decontamination of saffron by different postharvest processes

Saffron is transported as a dried product, and production processes such as growth, harvesting, drying, storage, transportation, grinding, and handling occurs in limited countries. Saffron, as a spice, is often used in small amounts and the presence of any living contaminant will inevitably be eliminated during the cooking process, so the main problem is the contribution of contaminants to spoilage of the product leading to undesirable changes in flavor and taste. However, in global saffron marketing, poor quality could be critical especially during transportation (Zhang et al., 2012).

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A study on the quality parameters and shelf stability of saffron demonstrate that the short harvest periods will result in keeping quality parameters. Decontamination of microorganisms in herbal plants is usually done with heat treatment, irradiation with gamma rays, or electron beam and ozone (Akbari et al., 2014). Commercial and legal requirements regarding the safety, quality, and storage of food products have focused on the development and improvement of decontamination methods.

19.5.1 Effect of drying processes on microbiological quality of dried stigma Saffron is very hygroscopic, and exposure to moisture creates a risk of spoilage of the product. Before drying, the relative humidity of saffron is about 70%, while the water content is 9%
10% (dried final product) and 12%
18% (wooden packaging). The postharvest treatment of drying stigmas is necessary to reduce the moisture to a value below 15% and to obtain a microbiologically stable product (Naviglio et al., 2010). Generally, the dehydration methods are classified as traditional and industrial drying.

19.5.1.1 Traditional drying Open sun and shade drying are the traditional methods still widely used because of their simplicity and low cost. These drying methods are still used in Iran, India, and Morocco for saffron stigma drying. In sun drying, stigmas are dried for 3
5 days in the sun until the moisture content is reduced to 10%
12%. Alternatively, during flower harvest, the whole flowers are dried in the sun and stigmas are picked by hand from the dried flowers later (Aghbashlo et al., 2013). The dried, warm saffron is then allowed to cool in a dry place (Celma et al., 2008). This traditional way of drying that has uncontrolled conditions has adverse effects on the quality of saffron. Sampathu et al. (1984) noted that during the long periods of traditional drying, enzymatic activity is high and microbial pollution occurs. Thus, a shorter drying time and controlled temperature will result in higher-quality products.

19.5.1.2 Artificial drying Artificial drying methods are carried out at higher temperatures and have been employed in saffron processing in countries such as Spain, Greece, and Italy (Carmona et al., 2005). In spite of some of the advantages of industrial dryers, including achievement of hygienic conditions, quality control, and reduction of product loss and process duration, the energy requirements of this drying technology are high. The drying process also releases carbon oxides to the environment. Therefore, it is crucial not only to assure good quality of the dried products but also high-energy efficiency and low environmental impact (Aghbashlo et al., 2013). Milani et al. (2016) introduced a strategy for monitoring microbiological criteria of saffron including hazard identification and risk assessment during harvesting, transportation, separation, and also drying. Samples were collected from a typical farm in Kashmar, Iran. It is recommended to choose blooms rather than saffron flowers. For this purpose, the laborers wear gloves to pick the blooms to maintain good hygiene, then transport and keep the produce in cool conditions at 4 C before treatment. Stigmas for the experiments were separated by aseptic tweezers under laminar flow cabinet. Dried stigma was obtained by electrical heater (Spanish method) at 55 C for 45 minutes until they reached the desired moisture content. All laboratory procedures were carried out under sterile conditions and microbiological count determined. Microbial analysis of S. aureus, total bacteria count coliform, E. coli, mold, and yeast was carried out the day after arrival. The results are given in Table 19.1.

19.5.1.3 Freeze-drying process Freeze drying, also known as lyophilization, is a separation process widely used in biotechnology, fine chemicals, and the food and pharmaceutical industries (Atefi et al., 2004). Heat-sensitive materials, fine chemicals, biotechnological products, and some pharmaceuticals, which might lose their quality in conventional evaporative drying, can be safely freeze dried (Mazloumi et al., 2007). The freeze-drying process is a multistage, relatively slow, and expensive process due to the initial investment and high operating costs (Acar et al., 2011). Some researchers have reported that the long drying time and high-energy consumption of the freeze-drying process can be lowered by the addition of microwave power to the conventional freeze dryer (Sadikoglu and Liapis, 1997; Wang et al., 2009). In order to obtain freeze-dried products with the highest quality, maintaining the stability of the product during each step of the freeze-drying process is critical and requires extreme caution. Substantially long drying time, intensive energy requirement, high initial investment cost along with labor and operating costs make freeze drying a very expensive separation process. Therefore, freeze drying is the method of separation of high market value products such as pharmaceuticals, fine chemicals, biotechnological products, and expensive foods (Acar et al., 2011; Wang et al., 2009). The temperature of the freeze dryer

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TABLE 19.1 Microbial analysis of saffron samples dried by different methods. Microbial analysis

Total microbial (CFU g21)

Coliform (CFU g21)

Mold (CFU g21)

Yeast (CFU g21)

Esherishia coli (CFU g21)

Vacuum oven

85 3 102

0

60

0

0

Freeze

65 3 10

1800

70

10

0

Solar

3 3 10

500

50

0

0

Microwave

67 3 102

30

20

0

0

Traditional

54 3 10

85 3 10

100

200

0

Drying method 3

3

3

2

From Mazloumi, A., Taslimi, S., Jamshidi, H., Atefi, H., Haj-Seyyed-Javadi, A., Komeili, P., 2007. Comparison of the effects of vacuum oven-, freeze-, solar-, and microwave-drying with traditional drying methods on the qualitative characteristics of Ghaen saffron. Iran. J. Nut. Sci. Food Technol. 2 (1), 69
76 (in Persian).

TABLE 19.2 Microbial quality of saffron samples produced under hygienic postharvest conditions. Microbial count

n

Total microbial activity $ 10 CFU

0

Mold and yeast $ 10 CFU

0

5

2

Enterobacteriaceae $ 10 CFU

0

Escherichia coli $ 101 CFU

0

3

Bacillus cereus $ 102 CFU

0

Clostridium perfringens $ 10 CFU 1

0

Clostridium sulfito-reductores $ 10 CFU 5

0

From Milani, E., Koocheki, A., Goliovahhed, Q.A., 2016. Assessment and monitoring of saffron microbiological criteria. Fifth International Symposium on Saffron Biology and Technology: Advances in Biology, Technologies, Uses and Market. 23
26 November 2016, Agadir, Morocco.

can be set in the range of 240 C to 260 C, while the condenser temperature can be set as low as 270 C. The vacuum pump of the freeze dryer is capable of supplying a vacuum as low as 15 Pa (Acar et al., 2011; Sadikoglu et al., 2006). Approximately 10 g of stigmas of the saffron flower are placed in the tray of the freeze dryer and the temperature of the plates are set to 240 C for 4 hours for complete freezing. The initial temperature of the heating plates of the freeze dryer is 230 C and increases gradually to 5 C, not causing any melting and scorching of the stigmas of the saffron. The drying chamber pressure is set to 50 Pa and kept constant during drying. The freeze-dried samples are packed with oxygen- and water-vapor-impermeable packaging material prior to chemical analysis (Acar et al., 2011). Atefi et al. (2004) evaluated the effects of the freeze-drying process on the quality parameters of saffron in comparison with traditional method. For this purpose, fresh saffron stigmas were immediately dried both in traditional conditions and freeze-drying conditions for 5 and 40 hours at 218 C, and chamber pressure was kept at 0.5 mmHg. Microbial analysis results indicated that E. coli was negative, and the number of coliforms, molds, and yeasts were low in freeze-dried saffron. Since dried stigma of saffron is a very high value material due to its application in foods, freeze drying can be used to produce saffron with acceptable quality. Mazloumi et al. (2007) compared the effects of different drying methods including vacuum oven, freeze, solar, and microwave drying with traditional drying methods on the qualitative characteristics of saffron produced in Ghaen, Iran. The results indicated that all four nontraditional methods of drying were better than the traditional method, although sun drying is recommended in rural areas (Table 19.2.)

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19.5.2 Effect of cold plasma process on microbiological quality of dried stigma Cold plasma is one of the most promising nonthermal decontamination techniques. The antimicrobial activity of cold plasma was investigated in a variety of foods. Researchers showed that A. flavous is eliminated after 11 minutes of plasma jet treatment (Amini and Ghoranneviss, 2016). The cold plasma treatment process is inexpensive as it is generated in atmospheric pressure and does not need a vacuum system. The input gas (Ar) is less expensive and any chemical or material is not required. Due to the antibacterial activity of cold plasma and its low cost, it could be used as an alternative technology for decontamination of saffron in large scales. Amini et al. (2017) studied the influence of cold plasma on crocin esters and volatile oils of saffron. The results showed that increasing the input voltage and increasing the amount of added oxygen to argon gas increased changes in the safranal and crocin esters. There was no trans-2G, cis-4GG, or cis-3Gg compounds observed after the Ar/10% O2 cold plasma treatment at 12 kV. Nevertheless, there are no studies on changes on microbial quality of compounds after cold plasma treatment and studies on changes in food compounds after cold plasma treatment are rare.

19.5.3 Effect of ozone treatment on microbiological quality of dried stigma Selecting a suitable method for disinfecting medicinal plants is very important. Today, ozone is one common method of disinfecting plant material that is chosen over other methods. The Environmental Protection Agency (EPA) have confirmed that ozone is safer than chlorine and other disinfectants (Selma et al., 2008). Ozone treatment is replacing chlorine (Kim et al., 1999) due to its high oxidizing properties (Perry and Yousef, 2011). The ozone as a treatment of postharvest in vegetables and fruits are used as gas and water (Oztekin et al., 2006; Selma et al., 2008; Zeynep et al., 2004). The important point about utilization of ozone as a disinfectant is about 20 minutes half-life of ozone in water at room temperature and when it decomposes into simple molecules of oxygen, which is not harmful for consumers (Kim et al., 1999). There is limited information on the effect of ozone on microbial decontamination of medicinal plants. The effect of ozone on microbial inactivation of red pepper was examined by Akbas and Ozdemir (2008). The results showed that ozone treatments with concentration of 1 mg L21 for 360 minutes can reduce the number of E. coli and B. cereus and ozone concentrations greater than 5 mg L21 can be used for the reduction of B. cereus spores. Akbari et al. (2014) evaluated microbial decontamination of saffron by ozone treatment. The aim of the study was to omit or reduce the population of pests and microorganisms without any noticeable damage to the color, flavor, and fragrance of saffron. For the purpose of inactivating microbiological contamination of saffron, an ozone generator, which produced 5 g h21 ozone, was used. Four levels of ozone for 0, 1, 2, and 3 hours were used. Then microbiological tests including total count of bacteria, coliform, mold, and yeast and chemical analyses of the main characteristics of saffron were carried out. Moreover, the samples were examined for the presence of living larvae, after using 4.7 ppm dosage of ozone. The results showed that in the fourth level of ozone where the input of pure oxygen to the ozone generator was 6 L min21 for 3 hours contact time of saffron with ozone, the number of total count of bacteria, coliform, mold, and yeast decreased up to 93.3%, 99.8%, 96.9%, and 84.5%, respectively, while the crocin, saffranal, and picrocrosin estimation showed a decrease of 14.9%, 10.46%, and 13.85%, respectively. The use of ozone (4.7 ppm) for 20 minutes annihilated almost 84% of larvae whereas after further ozonation exposure for 40 minutes almost no larvae remained alive. This study suggests the possibility of using ozone as an agent for saffron decontamination.

19.5.4 Effect of irradiation treatment on microbiological quality of dried stigma 19.5.4.1 Microwave treatment Microwaves (MW) are defined as electromagnetic waves in the range of infrared and radio waves, with a wavelength ranging from 1 mm to 1 m and operating at a frequency of 300 MHz
300 GHz (Thostenson and Chou, 1999). In the United States, two frequencies of 915 and 2450 MHz are designated by federal regulations for industrial application (Lau and Tang, 2002). It seems natural therefore to adapt MW radiation to pasteurize or even sterilize foods at low temperatures in shorter times than required by conventional methods (Akgul et al., 2008). Despite some disadvantages, such as nonheterogeneous heating treatment, edge overheating, soggy texture, and browning, MW processing can be considered as an alternative approach to food stabilization due to the possibility of treating the product inside the packaging (Brody, 1992). Processing of spices using MW is a newer technique, but it has shown to be preferred due to its convenience and ease of handling (Behera et al., 2004). It is well known as an effective treatment against a wide range

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of microorganisms in various food systems, including E. coli (Can˜umir et al., 2002), S. aureus (Yeo et al., 1999), Bacillus spp. (Celandroni et al., 2004), Campylobacter jejuni (Uradzinski et al., 1997), Pediococcus spp. (Kozempel et al., 1997), Saccharomyces cerevisiae, and Lactobacillus plantarum (Tajchakavit et al., 1998). Bacterial decontamination of food products after exposure to MW irradiation has been well documented (Akgul et al., 2008; Basaran, 2009). Aydin and Bostan (2006) studied microbial decontamination of powdered black pepper (Piper nigrum L.) by using MW. An innovative MW process for microbial decontamination of spices and herbs was introduced by Dababneh (2013). Based on experimental results, the suitability of MW for decontamination of spices and herbs in less than 60 seconds at 900 W and it could be used at home and at industrial levels. Comparison of MWassisted drying of saffron with traditional method was studied by Hosseiny-Nejad et al. (2002). Using the traditional Iranian method, around 20 g of fresh stigmas were dehydrated by spreading them on a piece of paper at room temperature for 4 days. The dehydration of 5 g samples of fresh stigmas was carried out at three different powers (200, 600, and 1000 W) using a National Model microwave. In drying with an electric oven, 5 g of saffron stigmas were placed in Petri plates at dried at temperatures of 55 C, 65 C, and 85 C). The results showed that MW-dried samples had significant difference with samples dried in traditional method.

19.5.4.2 Electron beam and gamma irradiation treatment Application of ionizing radiate treatment of food on an industrial scale began at the beginning of the 1980s after the Joint FAO/IAEA/WHO Expert Committee accepted. Irradiation of food at a dose level of 10 kGy or below is toxicologically safe and nutritionally adequate (Byun et al., 2008; Kim et al., 2009). Food irradiation with ionizing radiation was introduced as an easy and reliable technological process for reducing spoilage losses, improving hygienic quality, and extending shelf-life (Kim et al., 2009; Lim et al., 2007). The recommended dose levels are low at 1 kGy to inhibit insect infestation and delay ripening; medium at 1
10 kGy to reduce bacterial load (particularly pathogens); and high at 10
50 kGy for commercial sterilization and elimination of viruses (Chau et al., 2007). Ionizing radiation is a method for preservation of foods that uses the high energy of gamma rays or accelerated electrons, thereby ionizing molecules (Chau et al., 2007). Spice irradiation is treatment with radiant energy to obtain some beneficial effects including disinfestation, improvement of shelf-life by inactivation of spoilage organisms, and improvement of the safety of spices by inactivating foodborne pathogens. Ray irradiation is now internationally recognized as an effective method for maintaining the quality of spices for a long time. Ghoddusi and Glatz (2004) studied the decontamination of saffron by electron beam irradiation. For this purpose, the dried stigmas of saffron were inoculated with three levels of 103, 104, and 106 CFU g21 of a mixed culture of bacteria, yeast, and mold isolated from the natural contaminant flora of saffron, and were irradiated at four dose levels (2, 5, 10, and 15 kGy) in an electron beam irradiator. Yeasts were most resistant to irradiation; some survivors were found even at doses as high as 15 kGy. Molds and bacteria were less resistant and were eliminated at 5 and 10 kGy, respectively. The calculated D-value for the mold, bacterium, and yeast used were 0.82, 0.86, and 2.69 kGy, respectively. Jouki et al. (2011) evaluated the effects of gamma irradiation for improvement of saffron shelf-life. Samples were treated with 0 (none irradiated), 1.0, 2.0, 3.0, and 4.0 kGy of gamma irradiation and held for 2 months. Control and irradiated samples underwent microbial analysis and chemical characteristic and sensory evaluation at 30-day intervals. The results indicated that an irradiation dose of 3 kGy can effectively control microbial growth in saffron and extend shelf-life without any significant deterioration of quality constituents. This technology will enable food processors to deliver larger amounts of high-quality saffron with longer shelf-life.

19.5.5 Infrared irradiation treatment IR radiation is an increasingly popular method used to dry moist materials. It is known as an artificial drying method that increases the moist material temperature and evaporates its moisture by IR wavelength radiation from a source that interacts with the internal structure of the product. IR heating has advantages such as decreasing drying time, highenergy efficiency, and lower environmental impact. The energy of radiated waves is transferred from the source to the sample product without heating surrounding air leading to higher temperature in the inner layers of the samples compared to the surrounding environment and more heat transfer (Celma et al., 2008). Torki-Harchegani et al. (2017) assessed the quality characteristics of saffron stigmas affected by IR thin-layer drying. Drying temperatures were fixed at 60 C, 70 C, and 110 C, and dehydration of the samples occurred in a short

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accelerating rate period at the start followed by a falling rate period. Analysis of the results confirmed that room temperature need a longer drying times of 4 days, therefore poor quality material would be produced. Saffron samples at an optimum temperature of 80 C
90 C in IR drying and 65 C
75 C in oven drying contained the highest amount of crocin after drying). To the best of our knowledge, no information on microbial decontamination of saffron by IR drying is available in the literature.

19.6

Saffron packaging

After harvesting, separation, and drying processes saffron is usually packaged in well-sealed containers and stored in cool places. However, long-term storage, especially in moist and warm places, may make the product prone to reabsorption of water and regeneration of contaminating microorganisms and finally putrefaction of the saffron. A package is a manufactured product consisting of any material or a combination of materials that can be used to present, contain, protect, handle, and distribute goods from raw materials to finished products in every phase of the distribution chain. Numerous plastic polymers with different barrier properties are commercially available (Mills et al., 2012). Dried stigma should be properly packaged and stored far from moisture and light at temperatures between 5 C and  25 C. According to the Institute of Standards and Industrial Research of Iran (ISIRI): 5689 document, enterococcus species and E. coli should not be present in saffron products at all, and the maximum permissive levels of spore-forming sulfite-reducing clostridia and molds in one gram of saffron should be limited to less than 102 and 103 microorganisms, respectively (Ahari et al., 2013). In addition to the above mentioned microorganisms, European regulatory bodies have strictly recommended analysis of saffron samples for identification of other species, including B. cereus, salmonella species, and C. perfringens (Cosano et al., 2009). Karazhiyan et al. (2012) investigated the microbial properties of dried stigma during 12 months after harvesting. In their study, stigmas were separated and the samples dried in sieve by electrical heater according to the Saffron National Standard of Iran. A second sorting was done, and then the samples were packed in polyethylene films and stored at 20 C
25 C in the lab. The results showed that moisture content and total count of saffron decreased during storage. Analysis of data indicated that the largest variations during the year of storage occurred from the beginning of harvesting time to 8 months after storage and after that variations were not statistically different.

19.6.1 Modified atmosphere packing Modified atmosphere packing has been used for increased distribution range and longer shelf-life. The effects of the gases normally used in modified atmospheres (O2, CO2, and N2) have been extensively studied (Church, 1994; Jeremiah and Gibson, 2001). The effects of different packaging methods of air and modified atmosphere packaging combined with irradiation (0.0, 1.0, and 2.0 kGy) on the preservation of saffron samples stored at room temperature for up to 60 days were investigated by Jouki and Khazaei (2013). In this study, microbial analysis of aerobic bacteria, coliform, E. coli mold, and yeast was carried out. Among the analyzed bacteria, coliforms were most sensitive to γ-radiation. The sensory and physicochemical analyses showed that the saffron samples packaged under modified atmosphere and irradiated with dose 2.0 kGy were acceptable under storage for 60 days, compared to 30 days for airpackaged nonirradiated samples.

19.7

Effect of antibacterial packaging on microbiological quality of dried stigma

There are many additive materials with antimicrobial or sensory properties that can be easily added to plastic before polymerization without any changes in the physical properties of the final composite. Flexibility, clarity, low cost, ease of transport, storage, and use are well-known attributes of plastic (Mills et al., 2012). The films with antimicrobial activity could help to control the growth of pathogenic and spoilage microorganisms (Da silva et al., 2009; Kathiresan et al., 2009). Due to the outbreak of infectious diseases caused by different pathogenic bacteria and the development of antibiotic resistance, researchers are now searching for new antibacterial agents. Current advances in nanotechnology have brought about new facilities in the food industry with the introduction of numerous targeted packaging techniques. Adding nanocomposites or nanoparticles into packaging materials to ensure better protection of foods have emerged as novel antimicrobial agents and the unique chemical and physical properties (Chau et al., 2007; Rai et al., 2009). Potential benefits for consumers and producers of these new products are widely emphasized (Bouwmeester et al., 2009).

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Antibacterial and antifungal properties of silver ions have already been recognized (Feng et al., 2000; Yamanaka et al., 2005). Bulk silver is chemically inert, and rarely releases Ag 1 ions into solution. In contrast, researchers have emphasized that silver nanoparticles (AgNPs) have shown notable chemical activity compared to bulk silver metal due to the higher specific surface area-to-volume ratio of AgNPs the resultant antimicrobial effects of Ag-NPs are higher 10
12 (Echegoyen and Nerin, 2013; Lee et al., 2012; Reidy et al., 2013). Sales et al. (2012) reported after a while, 11%
49% of total Ag content of silver nanoparticles as Ag 1 ions was released into the solution. Considering the side effects of antibiotics and the occurrence of antibiotic resistance in Pseudomonas aeruginosa and S. aureus, the use of natural agents has attracted the attention of scientists. Cushen et al. (2014) reported 0.1%
0.5% filling rate of composites analyzed for exploring silver migration into solutions. The composites usually are used for packaging liquid products not for dry crops like saffron. As mentioned earlier, the antibacterial effects of AgNP composites were delayed due to the dry nature of samples compared to soluble products like beverages and drinks. AgNPs as Ag ions may easily be released into soluble environments and its release rate is mainly under control of several factors including moisture content of the product, time of contacts between NP and the environment, pH and temperature of the product, presence of oxygen or other oxidoreductants (Echegoyen and Nerin, 2013). That means Ag 1 release from AgNPs are conversely related to pH value and high release occurs in lower pH solution (Barrena et al., 2009; Cho et al., 2005; Song et al., 2011; Von Goetz et al., 2013). The findings showed that 10 minutes exposure of E. coli cells to a small piece of AgNP-4000 ppm composite immersed in liquid media resulted in complete elimination of the bacteria from the solution. That is why spiked saffron samples in the study did not become sterile at all, even after 6 months storage in relation with 4000 ppm silver composites. Ahari et al. (2013) also reported such findings and Ag 1 release in the study from composites containing 4000 ppm AgNPs into products were equal to zero. The effect of polymer/nanosilver composite packaging on the long-term microbiological status of Iranian saffron was evaluated by Eslami et al. (2016). Identification processes were carried out according to the ISIRI: 9433 for sulfitereducing bacteria under anaerobic conditions, ISIRI: 1810 for salmonella species identification, ISIRI: 10899-2 for fungi species, ISIRI: 2198 for enterococcus species, ISIRI: 2946 for E. coli, ISIRI: 10530 for B. cereus, ISIRI: 2197 for C. perfringens, and ISIRI: 6806-3 for coagulase-positive staphylococci. The results showed that 4000 ppm nanosilver composites (0.4% fill rate) had the best function, but the effect was very weak and associated with several months’ delay, probably due to lacking a suitable bed for ion migration or for AgNP movement. Composites with different nanosilver concentrations are commercially available. Samiee et al. (2017) investigated the influence of the antibacterial activity of AgNPs and methanolic extract of saffron on some bacterial strains. The results showed that the combination of medium concentrations of Ag-NPs (500 μg mL21) and saffron extract (50 mg mL21) was in the optimum mode to eliminate S. epidermidis and S. pyogenes. The results also showed that saffron extract, AgNPs, and their combined form had antibacterial effects on these bacteria. The synergistic effects of active components of the extract and antimicrobial preservatives used in food, health, pharmaceutical, and cosmetic industries should be evaluated.

19.8

Effect of Hurdle technology on microbiological quality of dried stigma

Hurdle technology is more effective than irradiation or AgNP packing methods when they are used alone. Therefore, a combined method could be used for microbial decontamination of saffron with no significant differences on chemical characteristics and sensory attributes. Gamma irradiation and AgNPs have positive effects on preventing decay by sterilizing microorganisms and improving safety without compromising the nutritional properties and sensory qualities of foods (Ahn et al., 2004; Oh et al., 2005). Sales et al. (2012) investigated the combination effects of gamma irradiation and AgNP packaging on the microbial contamination of saffron during storage. For this purpose, saffron samples were packaged by polyethylene films with up to 300 ppm nanosilver particles as antimicrobial agents and then irradiated in cobalt-60 irradiator (gamma cell Model: PX30, dose rate 0.55 Gry/second) to 0, 1, 2, 3, and 4 kGy at room temperature. For each dose of gamma irradiation three samples were used. This procedure is commercially used for irradiation of prepackaged spices. Two samples were analyzed immediately after irradiation and subsequently at regular intervals during storage (1, 30, and 60 days) at ambient temperature. The results showed that the optimum gamma irradiation dose to decrease total aerobic mesophilic bacteria to zero was 4 kGy for Polyethylene (PE) film without nanosilver particles. But it was 3 kGy for saffron samples packaged by PE film with nanosilver particles. Irradiation of the saffron samples packaged by PE films with nanosilver particles showed the best results for decreasing microbial contamination at 2 kGy and for PE films without silver

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nanoparticles it was 4 kGy. Finally, it can be concluded that the combined method of gamma irradiation and packaging with nanosilver particles has more positive effects on microbial safety.

19.9

Conclusion

Microbiological quality is a major concern in the food industry because of the acute risk to health posed by bacteria, mold, and yeast. Concerns have been raised repeatedly regarding the contamination of saffron. Postharvesting is a critical control point with major effects on the chemical and microbiological characteristics of saffron. Prevention of microbial contamination in dried stigma requires good hygiene practices and hazard analysis and critical control points (HACCP) at all stages of production. This chapter provided data concerning the contamination of saffron and microbiological standards put in place to prevent it. Maintaining good microbiological quality of saffron is a key priority for both saffron suppliers and consumers.

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Further reading Benzi, G., Ceci, A., 1997. Herbal medicines in European regulation. Pharmacol. Res. 35, 355
362. Diehl, J.F., Josphson, E.S., 1994. Assessment of the wholesomeness of irradiated food. Acta Aliment. 23, 195
214. Kim, K.H., Yook, H.S., 2009. Effect of gamma irradiation on quality of kiwifruit. Radiat. Phys. 78, 414
421. Sadikoglu, H., Ozdemir, M., Seker, M., 2003. Optimal control of the primary drying stage of freeze drying process in vials using variational calculus. Dry. Technol. 21, 1307
1331. Sagoo, S.K., Little, C.L., Greenwood, M., Mithani, K.A., Grant, J., McLauchlin, E., et al., 2009. Assessment of the microbiological safety of dried spices and herbs from production and retail premises in the United Kingdom. Food Microbiol. 26, 39
43. Sospedra, I., Soriano, J.M., Man˜es, J., 2010. Assessment of the microbiological safety of dried spices and herbs commercialized in Spain. Plant Food Hum. Nutr. 65, 364
368. WHO, 1999. High-Dose Irradiation: Wholesomeness of Food Irradiated With Doses Above 10 kGy. Report of a Joint FAO/IAEA/WHO Study Group, WHO Technical Report Series 890. World Health Organization, Geneva, Switzerland.

Chapter 20

Saffron adulteration Arash Koocheki1 and Elnaz Milani2 1

Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran, 2Food Science and

Technology Research Institute, ACECR, Mashhad, Iran

Chapter Outline 20.1 Introduction 20.2 Detecting adulteration 20.2.1 Chromatographic techniques 20.2.2 Infrared spectroscopy 20.2.3 1H nuclear magnetic resonance

20.1

321 321 322 325 328

20.2.4 Molecular techniques 20.2.5 Electronic nose 20.3 Conclusion References

328 331 332 332

Introduction

The spice trade has one of the longest and richest histories of any industry. Among spices saffron is considered one of the most valuable due to its ability to add color, taste, and aroma to various foods. Global consumption of saffron is 300 tons and the world trade of saffron is worth 1 billion dollars, which accounts for 3%
4% of the total spices sold internationally (Islamic Republic News Agency). Fraud in the food industry has a long history and dates back thousands of years. Saffron is considered as one of the most common target of fraud (Negbi, 1999). This is mainly due to the high price of the spice and to the lack of technological methodologies available to detect fraud. Fraudulent practices are most common when the spice is in its powdered form. The history of fraudulence in saffron has been reported as far back as 600 years ago. In those days, serious punishment was imposed for fraud. In some European countries such as Italy, special armed police were established to defend the authenticity of this spice. The addition of a variety of less expensive and more readily available plant materials, mostly of inferior quality but similar in appearance, has been a common fraudulent practice throughout history. The term adulteration refers to the addition of mineral substances, oils, or molasses for increasing weight; it can also refer to the addition of various dye material to improve its appearance. Addition of artificial colorants is a common way of adulteration with the aim of improving the appearance of dried stigmas or other extraneous materials to misealder consumers. As a general rule, artificial colorants should be absent from saffron according to the ISO 3632 standards (ISO, 2011, 2010). The most commonly occurring adulterations in saffron are listed in Table 20.1 (Sforza, 2013). Besides synthetic dyes, saffron powder can also be adulterated by addition of natural colorants from other plant tissues. Unlike the stigmas, saffron flower petals are not used commercially and are often used for adulteration of saffron powder. Red table beet, which is rich in betanine, and safflower, which contains carthamidin and carthamin are also used as adulterants. Finally, saffron can also be adulterated by the addition of madder, which is characterized by reddish hydroxyanthraquinones.

20.2

Detecting adulteration

Saffron quality is of major importance to most consumers. Therefore, for the purposes of saffron trading, evaluation of the purity and authenticity of a given product or grading its quality is important. The amount of crocin, picrocrocin, and safranal, which are responsible for saffron color, flavor, and aroma are used to determine the quality of saffron (Fernandez, 2004). The higher the amounts of these compounds, the higher the quality of the saffron. Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00020-4 © 2020 Elsevier Inc. All rights reserved.

321

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TABLE 20.1 Most common forms of adulteration in saffron and saffron powder. Forms of adulteration

Adulteration consisting of

Without the addition of foreign substances

Mixing with condensed or older saffron

Adding various parts of the saffron plant

Adding stamens or cut and colored perigone

Adding substances that increase weight

1. Increase humidity percentage 2. Soaking in sirup, honey, glycerin or olive oil 3. Adding chemicals such as barium sulphate, sodium, calcium, calcium carbonate, potassium hydroxide, potassium nitrate, monopotassium tartrate, sodium borate, lactose, starch, or glucose to the above sirups

Adding parts from other plants

1. Carthamus tinctorius flowers 2. Calendula officinalis flowers 3. Stigmas from other saffron types that are shorter and have no dye properties (Crocus vernus, Crocus speciosus, etc.) 4. Papaver rhoeas L., Punica granatum, Arnica montana, Scolimus hispanicus flowers cut in slices. 5. Perianths from certain spices such as carnations 6. Ground red pepper 7. Herbaceous plants cut in pieces and colored in azoic dye substances 8. Small allium porrum roots 9. Sandalwood and campeche wood powder 10. Curcuma

Adding animal substances

Salted and dried meat fibers

Adding artificial substances

Colored gelatin fibers

Adding organic dye substances

Martins yellow, tropeolina, fucsina, picric acid, tartrazine, erythrocine, azorubine, Cochineal A red, orange yellow, naphtanol yellow, rocelline, red, etc.

Source: From Sforza, S. (Ed.), 2013. Food Authentication Using Bioorganic Molecules. DEStech Publications, Inc., Lancaster, Pennsylvania, USA.

Increasing concern of producers, retailers, and consumers for authenticity of valuable products such as saffron underlines the need for high-throughput analytical methods that allow, from a practical point of view, for the rapid screening of potential adulterations. Once the adulteration is detected and the adulterant identified, quantification is also required to estimate the level of adulteration (Petrakis et al., 2017).

20.2.1 Chromatographic techniques Different methods are used to detect adulteration. Chromatographic techniques are used to detect what is today the most common type of fraud—adulteration with water-soluble acid colorants. Saffron powder can often be adulterated by addition of synthetic dyes such as tartrazine, methyl orange, or ponceau-4R, which can be easily detected by LCDAD or LC-MS/MS. The thin layer chromatographic method allows the detection of artificial water-soluble dye acid substances. It applies to saffron threads as well as to saffron powder. The detected dye substances are yn oline yellow, S
napthol yellow, tartrazine, amaranth, A
cochineal red, azorubine, orange II, erythrocine, and rocceline. High Performance Liquid Chromatography (HPLC) method is used for the determination of three different elements: identification of dye substances responsible for saffron coloring intensity (crocines); identification of artificial dye substances, water-soluble acid, pursuant to the ISO/TS 3632, 2003; and identification of fat-soluble dye substances. Although usually accurate and reliable, a main drawback of chromatographic methods are that they are often timeconsuming and require expensive instrumentation. For this reason, in the last few years efforts have been focused on the development of quick and cheap assays for spice authentication, mainly based on UV-vis spectroscopy (ISO 3632-2) (Fig. 20.1). The adulteration can also be detected by chromatographic analysis coupled with a UV detector. As an example, Haghighi et al. (2007) applied a HPLC-UV analysis for the determination of the main saffron dyes in order to assess the possible addition of exogenous natural colorants (Fig. 20.2). The authors analyzed pure saffron before and after addition of known amounts of selected adulterants (saffron petals, madder, safflower, and red beet). The method obtained correct classification of pure saffron against those colored by addition of safflower, madder, and red beet. However, the method failed to recognize saffron colored by saffron petals.

FIGURE 20.1 UV-visible spectra of saffron (a, 0.01 mg mL21) and saffron petals (b, 0.25 mg mL21), safflower (c, 0.15 mg mL21), madder (d, 0.17 mg mL21), and red beet (e, 2 mg mL21) colorants. From Haghighi, B., Feizy, J., Kakhki, A.H., 2007. LC determination of adulterated saffron prepared by adding styles colored with some natural colorants. Chromatographia 66, 325
332.

FIGURE 20.2 Chromatograms of the methanol
water (50%, v/v) extract of the styles colored with the colorants of saffron petals (A), safflower (B), madder (C), and red beet (D) recorded at 520 (A), 402 (B), 260 (C), and 535 (D) nm, including 4-nitroaniline as internal standard (IS). Concentrations of IS and colored styles, 0.09 and 0.35 mg mL21, respectively. From Haghighi, B., Feizy, J., Kakhki, A.H., 2007. LC determination of adulterated saffron prepared by adding styles colored with some natural colorants. Chromatographia 66, 325
332.

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SECTION | IV Saffron processing

TABLE 20.2 HPLC/PDA/MS chemical characterization of saffron, safflower, marigold, and turmeric extracts. Peak

Name

Rt (min)

UV-vis (nm)

[M
H]2 (m/z)

Crocus sativus L. (saffron) 1

4-(α-D-glucopyranosyl)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (picrocrocin)

20.2

250

375

2

trans-Crocetin (β-D-neapolitanosyl)-(β-D-gentibiosyl) ester

41.2

260, 440

1137

3

trans-Crocetin di-(β-D-gentibiosyl) ester

46.5

260, 420, 460

975

4

trans-Crocetin (β-D-glucosyl)-(β-D-neapolitanosyl) ester

50.1

260, 440

975

5

cis-Crocetin (β-D-glucosyl)-(β-D-gentibiosyl) ester

51.6

260, 330, 435, 460

813

6

cis-Crocetin di-(β-D-gentibiosyl) ester

57.5

260, 320, 435, 460

976

7

cis-Crocetin di-(β-D-glucosyl) ester

58.8

260, 325, 440, 465

813

Calendula officinalis (marigold) a

Quercetin 3-O-rutinosylrhamnoside

28.4

255, 355

755

b

Quercetin 3-O-rutinoside

30.6

255, 355

609

c

Isorhamnetin-3-O-rutinosylrhamnoside

32.2

255, 350

769

d

Narcissin

34.7

255, 355

623

e

Isorhamnetin 3-O-neohesperidoside

38.7

255, 345

623

f

Isorhamnetin-3-O-glucoside

39.9

255, 355

477

Carthamus tinctorius (safflower) I

Hydroxysafflor yellow A (safflomin A)

30.1

225, 410

611

II

6-Hydroxykaempferol 3-O-β-D-glucoside

30.6

275, 340

464

III

Kaempferol 3-O-β-rutinoside

37.7

265, 350

593

IV

Safflor yellow B

40.2

225, 410

1060

V

Anydrosafflor yellow B

42.1

225, 410

1044

VI

Prechartamin

58.7

240, 405

955

VII

Chartamin

68.6

370, 520

909

Curcuma longa (turmeric) α

Demethoxycurcumin

63.6

250, 425

337

β

Bisdemethoxycurcumin

63.9

250, 420

307

γ

Curcumin

64.3

260, 430

367

Source: From Sabatino, L., Scordino, M., Gargano, M., Belligno, A., Traulo, P., Gagliano, G., 2011. HPLC/ PDA/ESI-MS evaluation of saffron (Crocus sativus L.) adulteration. Nat. Prod. Commun. 6, 1873
1876.

The normative ISO 3632 (ISO 3632-1; ISO 3636-2) employed in the international trade market to determine saffron’s quality based on spectrophotometric and chromatographic measurements is clearly insufficient to assess saffron’s authenticity when saffron is adulterated with plant foreign matter with similar color and morphology. In fact, the ISO method is not able to detect adulterations with other plants such as safflower, marigold, or turmeric when their content is lower than 20% (Sabatino et al., 2011). High-performance liquid chromatography (HPLC) coupled with photodiode array (PDA) and electrospray ionization mass spectrometry (ESI-MS) detection has also revealed the addition of plant adulterants at a minimum of 2%
5% (w/w) (Sabatino et al., 2011). The HPLC/PDA/MS technique allowed the unequivocal identification of adulterant characteristic marker molecules that could be recognized by the values of absorbance and mass (Table 20.2). The selection of characteristic ions of each marker molecule revealed concentrations of up to 5%, w/w, for safflower and marigold and up to 2% for turmeric. In addition, the high dyeing power of turmeric allowed the determination of 2%, w/w, addition using exclusively the HPLC/PDA technique.

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325

FIGURE 20.3 LC-MS extracted ion chromatogram of glycosylated kaempferol derivatives and geniposide from an authentic saffron sample adulterated with 5% of gardenia extract in C18 (A) and cyano (B) columns. LC conditions: flow rate, 0.4 mL min21; mobile phases, water containing 0.1% formic acid (solvent A), and acetonitrile containing 0.1% formic acid (solvent B); elution gradient: 5%
17.5% B in 10 min; 17.5%
50% B in 2 min; 50% B for 4 min, 50%
5% B for 1 min, and 5% B for 10 min in order to reequilibrate the column at the initial conditions; injected volume, 5 μL; temperature 40 C. MS conditions in ESI 2 : capillary voltage, 3000 V; nozzle voltage, 0 V; drying gas conditions, 10 L min21 and 300 C; nebulizer pressure, 1.7 bar; sheath gas conditions, 6.5 L min21 and 300 C; fragmentator, 175 V; skimmer, 60 V; octapole voltage, 750 V. Peak identification: (1) Kaempferol 3,7,40 -O-triglucoside, (2) Geniposide, (3) Kaempferol 3-O-sophoroside-7-O-glucoside, (4) Kaempferol 3,7-O-diglucoside, (5) Kaempferol 3-O-sophoroside, and (6) Kaempferol 3-O-glucoside. (*) Different kaempferol derivatives not identified. From Guijarro-Dı´ez, M., CastroPuyana, M., Crego, A.L., Marina, M.L., 2017. A novel method for the quality control of saffron through the simultaneous analysis of authenticity and adulteration markers by liquid chromatography-(quadrupole-time of flight)-mass spectrometry. Food Chem. 228, 403
410.

A liquid chromatography-(quadrupole-time of flight)-mass spectrometry (LC-MS) methodology was developed by Guijarro-Dı´ez et al. (2017) to assess the authenticity of saffron through the analysis of a group of glycosylated kaempferol derivatives proposed as novel authenticity markers as a result of a metabolomic study of saffron (Fig. 20.3). A strategy was proposed to evaluate the minimum quantifiable adulteration percentage, which was established at 0.2% regardless of the adulterant employed. The determination of characteristic and endogenous compounds such as glycosylated kaempferols as authenticity markers could be a highly effective tool when detecting adulterations regardless of the adulterant employed. For example, the existence of a new adulteration method of saffron with gardenia is of interest to the developed methodology since it allows the detection of geniposide as an adulteration marker in saffron. The developed LC-MS methodology was successfully applied to the analysis of 19 commercial saffron samples through the analysis of glycosylated kaempferols and geniposide shown to be specific and suitable for the routine analysis because of its sensitivity, accuracy, and reproducibility. It is assumed that saffron authentication through established methodology is a challenging task, as saffron of higher quality may be intentionally blended with plant-derived substitutes to disguise fraud. Therefore, the development of analytical methodologies for reliable saffron quality control is of high interest for consumer protection and fraud prevention.

20.2.2 Infrared spectroscopy Infrared (IR) spectroscopy is often used as a simple, fast, and green method for the adulteration screening of botanical materials for foods and herbs. IR spectroscopy provides another versatile and cost-effective option for the high-

326

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FIGURE 20.4 Original MIR spectra of (A) saffron stigmas and (B) safflower petals. From Chen, J.-B., Zhou, Q., Sun, S.-Q., 2016. Adulteration screening of botanical materials by a sensitive and model-free approach using infrared spectroscopic imaging and two dimensional correlation infrared spectroscopy. J. Mol. Struct. 1124, 262
267.

FIGURE 20.5 Normalized synchronous 2D correlation MIR spectra of (A) saffron stigmas and (B) safflower petals in the range of 1500
1300 cm21. The autopeak spectra of (C) saffron stigmas and (D) safflower petals clearly show the autopeaks on the diagonals of the synchronous 2D correlation spectra. From Chen, J.-B., Zhou, Q., Sun, S.-Q., 2016. Adulteration screening of botanical materials by a sensitive and model-free approach using infrared spectroscopic imaging and two dimensional correlation infrared spectroscopy. J. Mol. Struct. 1124, 262
267.

throughput analysis of a diverse range of foods and herbs (Karoui et al., 2010). The increasingly recognized potential of portable/handheld IR spectroscopy renders this technique an effective fingerprinting tool (Ellis et al., 2012) for either laboratory or on-site analysis along complex supply networks. However, the overlapping of absorption signals of various substances significantly decreases the sensitivity and specificity of IR spectroscopy in the detection of adulterated samples. However, measuring the entities separately makes it possible to cluster the authentic and adulterant entities in a single sample. In comparison, near-infrared (NIR) spectroscopic imaging is more suitable for the measurement of plant samples, because most entities can be measured directly in the transmission or reflection mode. Therefore, NIR spectroscopic imaging can be used for the exploratory clustering analysis of the entities in plant samples. The applicability of IR spectroscopy for screening saffron adulteration with plant adulterants was also investigated by Chen et al. (2016) using IR spectroscopic imaging, 2D correlation IR spectroscopy, and principal component analysis (PCA) (Fig. 20.4). According to chemical compositions revealed by the original, second derivative, and 2D correlation mid-infrared (MIR) spectra, the cluster containing crocetin and crocins should be saffron, while the other cluster without crocetin or crocins should be the adulterant (Fig. 20.5). The feasibility of this approach was proven by the simulated

Saffron adulteration Chapter | 20

327

FIGURE 20.6 FTIR spectra of test set; (A) five samples from individual producers and (B) ten samples from blends of stigmas obtained from different producers. From Ordoudi, S.A., de los Mozos Pascual, M., Tsimidou, M.Z., 2014. On the quality, control of traded saffron by means of transmission Fourier-transform midinfrared (FT-MIR) spectroscopy and chemometrics. Food Chem. 150, 414
421.

adulterated sample of saffron. Accordingly, saffron adulterated by adding other plant materials can be detected by a simple, fast, sensitive, and green screening approach using IR spectroscopic imaging, 2D correlation spectroscopy, and necessary chemometrics techniques. However, information from both NIR microspectroscopic imaging and transmission Fourier transform infrared (FTIR) was required and only one adulterant (i.e., safflower) was evaluated. Fourier transform spectrometers have replaced dispersive instruments for most applications due to their superior speed and sensitivity. They have greatly extended the capabilities of infrared spectroscopy and have been applied to many areas that are very difficult or nearly impossible to analyze by dispersive instruments. Instead of viewing each component frequency sequentially, as in a dispersive IR spectrometer, all frequencies are examined simultaneously in Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy is a simple analytical technique largely applied for its rapidity and reproducibility in food fraud detection. FTIR spectroscopy was first employed for the characterization of crocins and related apocarotenoids in saffron (Tarantilis et al., 1998). The quality and authenticity issues of saffron using IR spectroscopic techniques have also been studied by Zalacain et al. (2005), Anastasaki et al. (2010), and Ordoudi et al. (2014). Transmission FTIR spectroscopy has also been used for detecting saffron adulteration or contamination with several colorants (Ordoudi et al., 2014) (Fig. 20.6). Karimi et al. (2016) reported the capability of FTIR spectroscopy combined with appropriate chemometric techniques to detect and quantify six different artificial colorants including tartrazine, Sunset yellow, Azorubine, Quinolone yellow, Allura red, and Sudan II in Iranian saffron. Analysis of the selected clusters of variables indicates that three regions band are responsible for differentiation of standard samples from their fraud ones. Their study showed that the combination of FTIR and clustering concept resulted in the best performance for calibration and external test set with 100% sensitivity and specificity.

328

SECTION | IV Saffron processing

Within the techniques used for measurements over the mid-IR region, where structural information related to the fundamental vibrations occurs (Lohumi et al., 2015), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) has proved its potential for the determination of quality and authenticity parameters of foods and herbs (Saltas et al., 2013). Petrakis and Polissiou (2017) presented an application of DRIFTS and chemometric techniques for evaluating adulteration of saffron with six characteristic adulterants of plant origin: Crocus sativus stamens, calendula, safflower, turmeric, buddleja, and gardenia. The proposed method involved a three-step process for the detection of adulteration as well as for the identification and quantification of adulterants. The results obtained illustrate that this strategy based on DRIFTS has the potential to complement existing methodologies for the rapid and cost-effective assessment of typical saffron frauds. The possibility of portable or handheld instrumentation may further enhance the potential of this approach for on-site analysis and fraud detection within long and complicated supply chains. Another advanced method, namely laser-induced breakdown spectroscopy, provides a rapid elemental analysis of the sample and can have application in food analysis (Bilge et al., 2015; Tiwari et al., 2013). FTIR and Raman spectroscopy are complementary methods that give molecular information about the sample, whereas LIBS identifies the elemental composition. Varliklioz et al. (2017) compared three spectroscopic techniques, namely, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Raman spectroscopy, and laser-induced breakdown spectroscopy (LIBS), and the superiority of the techniques was investigated by using PCA. Among these methods, LIBS was considered to be a promising tool for quantifying the level of adulteration in saffron samples with a simple and accurate process.

20.2.3

1

H nuclear magnetic resonance

Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique largely applied for its rapidity and reproducibility, having the potential for high-throughput analyses with minimal sample pretreatment (Longobardi et al., 2013). The development of NMR spectrometers with high-field magnets as well as advances in probe technology have enabled the analysis of numerous compounds in low concentrations, with high precision and accuracy (Bharti and Roy, 2012; Ohtsuki et al., 2013). NMR-based metabolite fingerprinting may identify the subtle differences that often exist between authentic and fraudulent products. This metabolomic approach has been explored to discriminate authentic saffron from commercial samples; the results indicated relative amounts of picrocrocin and the sum of different crocetin glycosides as the characteristic metabolites of authentic saffron (Yilmaz et al., 2010). The evaluation of adulteration with typical plant-bulking agents can be rapidly performed using 1H nuclear magnetic resonance (1H NMR) metabolite fingerprinting (Petrakis et al., 2015). This approach led to the development of reliable classification models for the detection and identification of adulterants utilizing the 1H NMR data obtained from the DMSO-d6 extracts of samples. Taking into account the deficiency of established methodologies to detect saffron adulteration with plant adulterants, the method reliably assessed the type of adulteration and could be viable for detecting saffron fraud at a minimum level of 20% (w/w). NMR metabolite fingerprinting has shown to be efficient for determining and identifying fraudulent additions of bulking agents to saffron, especially when plant adulterants are involved and the spice is commercialized in powder form. The obtained results confirmed the combined use of 1H NMR spectroscopy and multivariate data analysis as a valid and powerful tool to investigate quality and authenticity of food products. The use of advanced spectroscopic techniques such as 1H NMR may enable the rapid, nondestructive screening of quality and authenticity of a product with minimal or no sample preparation. Petrakis et al. (2017) used an NMR-based approach to identify and determine the adulteration of saffron with Sudan dyes (Fig. 20.7). They revealed that the high linearity, accuracy, and rapidity of investigation enable high-resolution 1H NMR spectroscopy to be used for evaluation of saffron adulteration with Sudan dyes. Accordingly, the main advantage of using NMR is the minimal sample preparation and null chemical treatment. In this respect, water-soluble artificial colorants, usually analyzed by other spectroscopic techniques, such as UV-vis and liquid chromatography, could be investigated as well, avoiding the drawbacks of sample pretreatment and the additional problems derived from the matrix effect that might influence the chromatographic resolution.

20.2.4 Molecular techniques The cheaper availability of biomolecular assays has made these DNA-related techniques affordable in a wide array of food-related applications, allowing the achievement of operating costs similar to those of UV detection (Dhanya and

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329

FIGURE 20.7 Aromatic region of 1H NMR spectra of Sudan I
IV dyes and the pure Greek saffron analyzed. From the bottom to the top pure saffron, Sudan I, Sudan II, Sudan III, and Sudan IV are represented. Specific signals for the identification of each Sudan dye in adulterated saffron are highlighted. From Petrakis, E.A., Cagliani, L.R., Tarantilis, P.A., Polissiou, M.G., Consonni, R., 2017. Sudan dyes in adulterated saffron (Crocus sativus L.): Identification and quantification by 1H NMR. Food Chem. 217, 418
424.

Sasikumar, 2010). As a consequence, DNA markers have become a popular means for the identification and authentication of a steadily increasing range of food products, spices, and medicinal or aromatic plants (Halima et al., 2007). Molecular markers can detect differences at the DNA level and offer numerous advantages over conventional phenotype-based alternatives because they are stable and detectable in all tissue types, regardless of the growth environment, development state, or differentiation status (Agarwal et al., 2008). The advantages of this approach include high sample throughput, low detection limit, and good interlaboratory reproducibility. As techniques develop, DNA-based methods could be used in the analysis of processed foodstuff, patent drugs, fixed paraffin, adequate extraction, or vegetable oil (Costa et al., 2012). Thus, molecular markers offer several advantages for the determination of food authenticity in routine quality control (Marmiroli et al., 2013). Currently, the majority of the methods developed for investigating saffron adulteration with bulking agents of plant origin rely on molecular techniques. Although very sensitive, such methods usually require extensive sample preparation and cannot easily address adulteration with plant extracts, probably due to the absence of recoverable DNA (Soffritti et al., 2016). Several PCR-based methods have been used to detect saffron adulteration, including allelespecific PCR (AS-PCR) (Mao et al., 2007), DNA sequence analysis (Ma et al., 2001), and RAPD-derived SCAR markers (Javanmardi et al., 2012; Marieschi et al., 2012). According to Marieschi et al. (2012) sequence-characterized amplified regions (SCAR) markers may represent a fast, sensitive, reliable, and low-cost screening method for the authentication of dried commercial saffron material. The method enabled the unequivocal detection of low amounts (up to 1%) of each adulterant, allowing the preemptive rejection of suspect samples (Fig. 20.8). Its enforcement limits the number of samples to be subjected to further evaluation with pharmacognostic or phytochemical analyses, especially when multiple batches have to be evaluated in a short time. AS-PCR and SCAR markers are derived from specific fragments. These markers require screening new fragments or alleles to design specific primers for any new or unfamiliar adulterants, which limits their application in the detection of new adulterants. Therefore, these techniques require time-consuming sequencing or have limited application. New molecular markers, particularly DNA barcode-based universal primers methods, have been proposed and are being rapidly developed (Jiang et al., 2014). To develop a straightforward, nonsequencing method for rapid, sensitive, and discriminating detection of these adulterants in traded saffron, Jiang et al. (2014) proposed a barcoding melting curve analysis method (Bar-MCA) that uses the universal chloroplast plant DNA barcoding region trnH-psbA to identify adulterants. Melting curve analysis is a fast and sensitive method for differentiating PCR production by fluorescence monitoring of the melting curve of the doublestranded DNA that is intercalated by the dye SYBR Green I in a real-time PCR system (Ririe et al., 1997). Results can be obtained without additional post-PCR processing in less than 2 hours. By amplification with the barcoding primer pair psbAF/trnHR and performing melting curve analysis, saffron was distinguished from the adulterants or detected in mixtures based on its melting temperature. This technique could detect the presence of an expected plant material and adulterant materials in close-tube reactions. Compared with other sequence-based species discrimination methods, melting curve analysis is a very promising technique, particularly in terms of costs and time. Villa et al. (2017) used an EvaGreen real-time PCR approach as a simple, fast, highly sensitive, and reliable method for the detection and quantification of safflower in saffron. Based on their research the normalized real-time PCR system could be used for the detection and quantification of even smaller amounts of safflower, since the technique enables positive amplification of the target down to 2 pg of DNA (B1.4 DNA copies) (Fig. 20.9). In this work, qualitative PCR

FIGURE 20.8 SCAR marker sensitivity assay: (A) PCR performed with ScAm190 primer pair specific for Arnica montana (1%, 2%, and 5% DNA from mixtures of C. sativus and A. montana). (B) PCR performed with ScBo267 primer pair specific for Bixa orellana (1%, 2%, and 5% DNA from mixtures of C. sativus and B. orellana). (C) PCR performed with ScCo390 primer pair specific for Calendula officinalis (1%, 2%, and 5% DNA from mixtures of C. sativus and C. officinalis). (D) PCR performed with ScCt131 primer pair specific for Carthamus tinctorius (1%, 2%, and 5% DNA from mixtures of C. sativus and C. tinctorius). (E) PCR performed with ScCv304 primer pair specific for Crocus vernus (1%, 2%, and 5% DNA from mixtures of C. sativus and C. vernus). (F) PCR performed with ScCl289 primer pair specific for Curcuma longa. (G) PCR performed with ScHsp354 primer pair specific for Hemerocallis sp. (1%, 2%, and 5% DNA from mixtures of C. sativus and Hemerocallis sp.). 2 , negative control, amplification with no template DNA; C.s., DNA from dried stigmas of C. sativus, as further negative control; A.m., DNA from dried flowers of A. montana, as positive control; B.o., DNA from dried seeds of B. orellana; C.o., DNA from dried flowers of C. officinalis; C.t., DNA from dried flowers of C. tinctorius; C.v., DNA from dried stigmas of C. vernus; C.l., DNA from dried rhizomes of C. longa (1%, 2%, and 5%), DNA from mixtures of C. sativus and C. longa; H. sp., DNA from dried tepals of Hemerocallis sp.; M, 100 bp DNA ladder. From Marieschi, M., Torelli, A., Bruni, R., 2012. Quality control of saffron (Crocus sativus L.): Development of SCAR markers for the detection of plant adulterants used as bulking agents. J. Agric. Food Chem. 60, 10998 2 11004.

FIGURE 20.9 Real-time PCR amplification with EvaGreen intercalating dye targeting the ITS region of safflower using binary reference mixtures [20%, 10%, 5%, 1%, 0.1% (w/w)] of safflower in saffron. (A) Amplification curves and (B) melting curves (targeting both ITS region of safflower and the eukaryotic gene). From Villa, C., Costa, J., Oliveira, M.B.P.P., Mafra, I., 2017. Novel quantitative real-time PCR approach to determine safflower (Carthamus tinctorius) adulteration in saffron (Crocus sativus). Food Chem. 229, 680
687.

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FIGURE 20.10 Specificity of the LAMP assay for the authentication of saffron and its adulterants. Turbidity was monitored with a Loopamp Realtime Turbidimeter at 400 nm every 6 s. 1 μL of calcein (a fluorescent detection reagent) was added to 25 μL of the LAMP reaction mixture before the LAMP reaction. Lines and tubes: 1, C. sativus 01; 2, C. sativus 11; 3, C. sativus 12; 4, C. sativus 13; 5, C. officinalis 01; 6, C. tinctorius 01; 7, C. longa 01; 8, D. carota 01; 9, N. nucifera 01; 10, Z. mays 01; and 11, Neg (negative control, ddH2O). From Zhao, M., Shi, Y., Wu, L., Guo, L., Liu, W., Xiong, C., et al., 2016. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence. Sci. Rep. 6, 25370. doi: 10.1038/srep25370.

and real-time PCR methods were proposed as specific, sensitive, accurate, and powerful tools for detection and quantification of safflower adulteration in commercial samples of saffron. Zhao et al. (2016) introduced a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants (Fig. 20.10). The LAMP method is a novel nucleic acid amplification technology that is quick, simple, and highly specific. LAMP is based on a complex methodology requiring four to six different primers that are specifically designed to recognize six to eight precise gene sequences. DNA amplification is accomplished by a DNA polymerase with strand-displacing activity, thus obviating the need for a thermal denaturation step to obtain single-stranded DNA (Notomi et al., 2015). The use of isothermal conditions in the LAMP technique allows for reactions to occur in less time because no temperature changes are required. This novel technique was sensitive, efficient, and simple for saffron adulteration detection.

20.2.5 Electronic nose Another study indicated the ability of an electronic nose system combined with PCA and artificial neural networks to differentiate nonadulterated and adulterated saffron (Heidarbeigi et al., 2015; Kiani et al., 2017). Saffron metabolic processes at the processing and storage duration make gases, which exist in the saffron aroma. It is therefore possible to determine the fake and original saffron by sensing these compounds in the headspace. An electronic nose is a device that identifies the specific components of an odor and analyzes its chemical makeup to identify it. An electronic nose consists of a mechanism for chemical detection, such as an array of electronic sensors, and a mechanism for pattern recognition, such as a neural network. Electronic noses include three major parts: a sample delivery system, a detection system, and a computing system. The sample delivery system enables the generation of the headspace (volatile compounds) of a sample, which is the fraction analyzed (Fig. 20.11). The system then injects this headspace into the detection system of the electronic nose. The sample delivery system is essential to guarantee constant operating conditions. The detection system, which consists of a sensor set, is the “reactive” part of the instrument. When in contact with volatile compounds, the sensors react, which means they experience a change of electrical properties. Heidarbeigi et al. (2015) used an electronic nose based on six metal oxide semiconductor sensors to detect the aroma fingerprints of saffron, saffron with yellow styles, and safflower and dyed corn stigma. They revealed that the system can recognize the saffron adulteration satisfactorily. The electronic nose was able to successfully differentiate nonadulterated and adulterated saffron at higher than 10% adulteration level. Advances and developments in sensor technology, chemometrics, and artificial intelligence make it possible to develop instruments based on artificial senses such as computer vision (CVS) and electronic nose (e-nose) systems

332

SECTION | IV Saffron processing

FIGURE 20.11 Schematic view of designed e-nose system. From Heidarbeigi, K., Mohtasebi, S.S., Foroughirad, A., Ghasemi-Varnamkhasti, M., Rafiee, S., Rezaei, K., 2015. Detection of adulteration in saffron samples using electronic nose. Int. J. Food Prop. 18, 1391
1401.

capable of measuring and characterizing color and aroma. Kiani et al. (2017) developed an integrated system based on CVS and electronic nose (e-nose) for saffron adulteration detection. Both CVS and e-nose techniques require little sample preparation and allow large data sets to be acquired in a short time. The CVS used was comprised of a CCD digital camera, standard lighting system, and software for image processing, which was also equipped with an e-nose system. They concluded that aroma characteristic variables were a little more effective than the color variables in detecting saffron adulteration.

20.3

Conclusion

Saffron authentication is a challenging task since saffron of higher quality may be blended with other materials to hide the fraud. Therefore, using accurate analytical methods to control saffron quality is of high interest for fraud prevention. Methods used to determine the saffron’s alteration based on spectrophotometric and chromatographic techniques are insufficient when saffron is adulterated with plants such as safflower, marigold, or turmeric. Saffron adulterated by the addition of other plant materials can be detected using infrared spectroscopy techniques. However, when using this method only one adulterant can be evaluated. Among the methods used for detection of saffron adulteration, NMR spectrometery with high-field magnets is an accurate and precise method for analysis of compounds in low concentrations. This method is reliable for assessing the type of adulteration and could be reliable for dealing with extensive saffron fraud at a minimum level of 20% (w/w). NMR metabolite fingerprinting is efficient for saffron powder especially when plant adulterants are involved. The majority of the methods used for investigation of saffron adulteration with bulking agents of plant origin rely on molecular techniques. These methods are very sensitive but usually require extensive sample preparation and cannot easily address adulteration with plant extracts. The LAMP method is a nucleic acid amplification technology that is quick, simple, and highly specific. Using saffron aroma in the headspace of the electronic nose is another possible method to determine fraudulent saffron. An electronic nose can identify the specific components of saffron odor and analyze its chemical makeup. Although several methods have been used to identify fraud in saffron, there is still no single method used to identify all the fraudulent in the samples.

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1: Saffron (Crocus sativus L.) Specification. The International Organization for Standardization, Geneva. Javanmardi, N., Bagheri, A., Moshtaghi, N., Sharifi, A., Kakhki, A.H., 2012. Identification of Safflower as a fraud in commercial Saffron using RAPD/SCAR. J. Cell Mol. Res. 3, 31
37. Jiang, C., Cao, L., Yuan, Y., Chen, M., Jin, Y., Huang, L., 2014. Barcoding melting curve analysis for rapid, sensitive, and discriminating authentication of saffron (Crocus sativus L.) from its adulterants. Biomed Res. Int. 2014, 1
10. Karimi, S., Feizy, J., Mehrjo, F., Farrokhnia, M., 2016. Detection and quantification of food colorant adulteration in saffron sample using chemometric analysis of FT-IR spectra. RSC Adv. 6, 23085
23093. Karoui, R., Downey, G., Blecker, C., 2010. Mid-infrared spectroscopy coupled with chemometrics: a tool for the analysis of intact food systems and the exploration of their molecular structure-quality relationships - a review. Chem. Rev. 110, 6144
6168. Kiani, S., Minaei, S., Ghasemi-Varnamkhasti, M., 2017. Integration of computer vision and electronic nose as non-destructive systems for saffron adulteration detection. Comput. Electron. Agric. 141, 46
53. Lohumi, S., Lee, S., Lee, H., Cho, B.-K., 2015. A review of vibrational spectroscopic techniques for the detection of food authenticity and adulteration. Trends Food Sci. Technol. 46, 85
98. Longobardi, F., Ventrella, A., Bianco, A., Catucci, L., Cafagna, I., Gallo, V., et al., 2013. Non-targeted 1H NMR fingerprinting and multivariate statistical analyses for the characterisation of the geographical origin of Italian sweet cherries. Food Chem. 141, 3028
3033. Ma, X.Q., Zhu, D.Y., Li, S.P., Dong, T.T., Tsim, K.W., 2001. Authentic identification of stigma Croci (stigma of Crocus sativus) from its adulterants by molecular genetic analysis. Planta Med. 67, 183
186. Mao, S.G., Luo, Y.M., Shen, J., Ding, X.Y., 2007. Authentication of Crocus sativus L. and its adulterants by rDNA ITS sequences and allele-specific PCR. J. Nanjing Norm. 30, 89
92. Marieschi, M., Torelli, A., Bruni, R., 2012. Quality control of saffron (Crocus sativus L.): development of SCAR markers for the detection of plant adulterants used as bulking agents. J. Agric. Food Chem. 60, 10998
11004. Marmiroli, N., Peano, C., Maestri, E., 2013. Advanced PCR techniques in identifying food components. In: Lees, M. (Ed.), Food Authenticity and Traceability. CRC Press, pp. 3
33. Moore, J.C., Spink, J., Lipp, M., 2012. Development and application of a database of food ingredient fraud and economically motivated adulteration from 1980 to 2010. J. Food Sci. 77, 118
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5. Ohtsuki, T., Sato, K., Furusho, N., Kubota, H., Sugimoto, N., Akiyama, H., 2013. Absolute quantification of dehydroacetic acid in processed foods using quantitative 1H NMR. Food Chem. 141, 1322
1327. Ordoudi, S.A., de los Mozos Pascual, M., Tsimidou, M.Z., 2014. On the quality control of traded saffron by means of transmission Fourier-transform mid-infrared (FT-MIR) spectroscopy and chemometrics. Food Chem. 150, 414
421. Petrakis, E.A., Cagliani, L.R., Polissiou, M.G., Consonni, R., 2015. Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by 1H NMR metabolite fingerprinting. Food Chem. 173, 890
896. Petrakis, E.A., Cagliani, L.R., Tarantilis, P.A., Polissiou, M.G., Consonni, R., 2017. Sudan dyes in adulterated saffron (Crocus sativus L.): identification and quantification by 1H NMR. Food Chem. 217, 418
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Section V

Economy and trade of saffron

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Chapter 21

Economic analysis of saffron production Naser Shahnoushi1, Leili Abolhassani1, Vida Kavakebi1, Michael Reed2 and Sayed Saghaian2 1

Department of Agricultural Economics, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran, 2Department of Agricultural

Economics, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, United States

Chapter Outline 21.1 21.2 21.3 21.4

Introduction World’s main exporters of saffron Price of saffron Saffron production around the world 21.4.1 Iran 21.4.2 India 21.4.3 Greece 21.4.4 Spain 21.4.5 Afghanistan 21.4.6 Summary 21.5 Production function in economics 21.5.1 Production function of saffron 21.6 Economic productivity of saffron 21.6.1 Water productivity of saffron 21.6.2 Labor productivity of saffron 21.6.3 Land productivity of saffron

21.1

337 337 338 339 340 341 341 342 342 343 343 344 344 345 345 345

21.7 Economic performance of saffron 21.7.1 Return on investment 21.7.2 Ratio of return to cost 21.8 Efficiency 21.8.1 Energy use efficiency 21.9 Economic comparative advantage 21.9.1 Revealed comparative advantage 21.9.2 Policy analysis matrix 21.9.3 Nominal protection coefficient 21.9.4 Effective protection coefficient 21.9.5 The domestic resource cost 21.9.6 Based on unit costs 21.10 Some economic advantages of saffron production 21.11 Conclusion References

346 346 346 346 346 349 349 349 352 352 352 353 353 353 354

Introduction

The purpose of this chapter is to examine and analyze the cultivation of saffron using simple economics to look at overall trends, followed by more technical analysis at the end. Most of the data provided for the analysis in this chapter are related to Iran. There are two reasons for this. One is that data and information related to other countries were not accessible for us. The second is that Iran, as we will see in the following section, is the major producer and exporter of saffron. We start this chapter by identifying the major exporters and describing the way saffron is priced, then we discuss saffron production in Iran. We end the chapter by applying some technical tools to analyze the economics of the saffron production process.

21.2

World’s main exporters of saffron

For many decades, Iran, Spain, and Greece have been the world’s major exporters of saffron. However, since 2000, the entry of other countries such as Afghanistan, China, the Netherlands, and Portugal into the world market has increased and influenced saffron export values. Fig. 21.1 presents the value of saffron exports from Iran, Spain, Greece, and Afghanistan during 2012 and 2016. Between 2012 and 2016, Iran and Spain held the first and second positions in saffron exporting. In most years, Greece has held the third position. In 2016 Afghanistan moved ahead of Greece into third place. Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00021-6 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 21.1 Exported values of the major exporting countries of saffron. Data from Trademap, 2017. Trade statistics for international business development. Available from: ,https://www.trademap.org/Index.aspx..

FIGURE 21.2 Price per kg of Iranian saffron in the world market (2011
17). Data from Trademap, 2017. Trade statistics for international business development. Available from: ,https://www.trademap.org/Index.aspx..

Iran is the main source of saffron in the world with 111,000 ha of saffron farms and about 404 tons of production in 2018 (Donya-e-Eqtesad, 2019). About 60% of the cultivated area is located in the three provinces of Khorasan (UNIDO, 2016). However, due to the lack of a packaging industry, proper marketing, and production of edible products with saffron, much of Iran’s saffron is sold to the world by other countries. The marketing process is that the saffron from Iran is sold to other countries, such as Spain, where it is appropriately packed and marketed to other countries at a high price.

21.3

Price of saffron

The price of saffron varies markedly by year. Fig. 21.2 shows the world price of Iranian saffron between 2012 and 2017. The highest price for saffron during the 2011
17 period was in 2011 at $2400 kg21. The lowest price was in 2012 at $1400 kg21. During 2013
16, the price of saffron remained relatively stable, but it jumped by $500 kg21 in 2016. The sudden drop in the Iranian saffron price in 2012 was mainly due to an increase in saffron supply. Fig. 21.3 shows the value of Iranian saffron exports. As can be seen, there was a dramatic increase in exports of saffron during 2008
12. Iran is the major producer of saffron, and while more than 90% of the world’s saffron is produced there, a significant portion of saffron’s value-add belongs to other countries due to Iran’s poorly developed processing sector (Karbasi, 2006). If Iran prioritized saffron processing in its development plans and suitably packaged saffron, it would be able to increase its value-add by more than 16 times (Resistance Analysts, 2016). Saffron price fluctuations not only influence producers, but also affect local economies where saffron is cultivated. According to a study conducted by Filipski et al. (2017) in the Taliouine
Taznakht region of Morocco, increasing saffron price by 77% over the years 2007
09 allowed improvements in production technology, such as drip irrigation,

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339

FIGURE 21.3 Export price of Iranian saffron (2000
15). Data from Trademap, 2017. Trade statistics for international business development. Available at ,https://www.trademap.org/Index. aspx..

TABLE 21.1 Saffron production in the world’s main saffron-producing countries in 2016. Yield (kg ha21)

Country

Area (ha)

Production (tons)

Share in the world (%)

Iran

105,270

336

3.3

88.8

India

5707

22

3.9

5.8

Greece

1800

7.2

4

1.9

Afghanistan

2811

6

2.1

1.58

3.2

0.68

Morocco

200

2.6

Spain

165

2.3

14

0.6

Italy

500

1

2

0.26

China

500

1

2

0.26

35

0.23

6.6

0.06

116,988

378.33

Azerbaijan Total




100

Source: Data from Iranian Ministry of Agriculture, 2017. Report of saffron. National plan of herbal medicine. Available from: ,http://si.torbath.ac.ir/ resource/file_book/8.pdf.; UNIDO, 2016. Saffron industry value chain development. Available from: ,https://open.unido.org/projects/IR/projects/120595..

which led to increased productivity of saffron flowers. This study showed that a 100% increase in saffron price brings a 133% increase in wages of a typical female employed for the harvesting stage, and a 36% increase in wages for a male employed for the cultivation stage. The price of saffron within the country, and most likely in the international market, is affected by its end use. Saffron used in food preparation and as a spice is more expensive than saffron used for medical purposes.

21.4

Saffron production around the world

As saffron grows under specific climate conditions, very few countries produce it and data are not available for all producing countries. Table 21.1 shows the area, amount, and yield of saffron production in the world’s main saffronproducing countries. Iran, Greece, Spain, and India have traditionally been the major saffron-producing countries (UNIDO, 2016). Fig. 21.4 shows the share of each country in world saffron production. Market shares by country have dramatically changed during the years, in particular after 2010, due to the increased demand for saffron. For instance, Afghanistan, which only started producing saffron in the late 1990s, accounted for 1.6% of world saffron production in 2016.

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SECTION | V Economy and trade of saffron

FIGURE 21.4 The share of Iran, India, Spain, and Greece in world saffron production. Data from UNIDO, 2016. Saffron industry value chain development. Available from: ,https:// open.unido.org/projects/IR/projects/120595..

FIGURE 21.5 The amount of saffron production in Iran during 2005
18. Data from Khorasan Razavi Agricultural directorate, 2019.

FIGURE 21.6 Saffron yield in Iran during 2005
18. Data from Khorasan Razavi Agricultural directorate, 2019.

21.4.1 Iran Saffron is a major agricultural product for Iran. Figs. 21.5
21.7 show the amount of production, yield, and land area during 2005
18. The year 2008 was the worst year in recent memory for saffron production in Iran. In that year, saffron production fell to less than 50,000 kg. The average yield was only 0.6 kg ha21 (the lowest in 10 years). The highest yield was obtained in 2005 at 4.1 kg ha21. Saffron yields since 2005 have averaged about 3.7 kg ha21. The land area of saffron cultivation has been increasing by about 6% annually since 2008.

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341

FIGURE 21.7 Saffron area in Iran during 2005
18. Data from Khorasan Razavi Agricultural directorate, 2019.

FIGURE 21.8 Cultivated area, amount of production, and yield of saffron in India during 1997
2017. Data from UNIDO, 2016. Saffron industry value chain development. Available from: ,https://open.unido.org/projects/IR/projects/120595..

21.4.2 India In India, most saffron is produced in the state of Jammu & Kashmir (Kafi and Showket, 2007). Fig. 21.8 shows cultivated area, production, and yield of saffron in India during the years 1997 and 2016. Reliable information for the years between 2005 and 2015 is lacking. India produces 15
20 tons of saffron per year but has tended to increase its production due to economic improvements. In order to protect its domestic consumption, the country has set a 35% tariff on imported saffron. In spite of the country’s intention to expand saffron farms, the cultivated area has only slightly increased from the 2010s—from 3143 ha in 2005 to 5707 ha in 2016. According to the evidence (UNIDO, 2016), Indian saffron is known as a lowquality product because of its high moisture content and dark color. Arab countries are the major exporters of saffron from India.

21.4.3 Greece Greece, with more than 5 and 7 tons of saffron production, was the second- and third-leading producer in 2004 and 2016, respectively. The major region of saffron cultivation in Greece is Kozani. The amount of saffron production in Greece, however, has markedly fallen since 2006 due to the country’s economic fluctuations and droughts. Fig. 21.9 presents cultivated area, production, and yield of saffron in Greece during the years of 2000
17.

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SECTION | V Economy and trade of saffron

FIGURE 21.9 Cultivated area, amount of production, and yield of saffron in Greece during 2000
17. Data from the Ministry of Agriculture, Greece, 2019.

FIGURE 21.10 Cultivated area, amount of production, and yield of saffron in Spain during 2000
11. Data from UNIDO, 2016. Saffron industry value chain development. Available from: ,https://open.unido.org/projects/IR/projects/120595..

After 5 years of significant reductions in Greece saffron production, the country has focused on expanding saffron cultivation. Some regions of Greece that abandoned saffron cultivation started to replant, and saffron cultivation increased by fourfold between 2010 and 2017.

21.4.4 Spain By 1970, Spain, with 60 tons of saffron production, was the leading producer. Cultivation, harvesting, and processing of saffron are very labor-intensive. Since 1990, production cost increases, particularly for wages, has decreased cultivation of saffron dramatically (Katawazy, 2013; UNIDO, 2016). For instance, production dropped to 3.4 tons in 2003, while it was 21.8 tons in 1990. Fig. 21.10 shows cultivated area, production, and yield of saffron in Spain in the years 2000
16. Since 2006, increases in the world price of saffron as well as the economic crises in Europe have encouraged farmers in Spain to cultivate saffron (UNIDO, 2016). In 2006, 116 ha of Spain lands were cultivated for saffron, while this area reached 165 ha in 2016. Spain, however, has the highest production yield in the world. In 2016, the average yields were 14 and 9 kg ha21 for irrigated lands and drylands, respectively. The saffron yield on irrigated lands is 19
20 kg ha21 (UNIDO, 2016).

21.4.5 Afghanistan In 1998, some Afghan NGOs in cooperation with local farmers started to produce saffron in the semiarid villages of Pashtun Zarghun district of Herat province. Saffron cultivation has expanded widely because of positive results. In 2002, the Ministry of Agriculture in Afghanistan began to distribute saffron corms to farmers in the provinces of Herat, Mazar-i Sharif, Baghlan, Kabul, Wardak, Bamyan, and Logar. Since 2003 the National Control Drug Strategy has been adopted, and saffron is expected to be an alternative cultivation to poppy (Wyeth and Malik, 2008; Mollafilabi et al., 2009).

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343

FIGURE 21.11 Areas cultivated (ha) by saffron in Afghanistan during 2004
16. Data from Katawazy, A.S., 2013. A comprehensive study of Afghan saffron. The report of Afghanistan Investment Support Agency (AISA); Iranian Ministry of Agriculture, 2017. Report of saffron. National plan of herbal medicine. Available from: ,http://si.torbath.ac. ir/resource/file_book/8.pdf..

FIGURE 21.12 Saffron yield production in Iran, Spain, and Greece during 2005
16. Data from UNIDO, 2016. Saffron industry value chain development. Available from: ,https://open.unido.org/projects/IR/projects/120595..

By 2011, more than 1300 farmers were growing saffron in these provinces (Katawazy, 2013; UNIDO, 2016). Fig. 21.11 shows the areas under saffron cultivation during the years 2004
16. According to Katawazy (2013), there are 7000
10,000 ha in Afghanistan that are suitable for saffron production and these areas could produce 50,000
70,000 kg of saffron.

21.4.6 Summary In summary, Iran’s share of saffron production has been decreasing since 2008. Newcomers with low wages, such as Afghanistan, have increased their share of production. Furthermore, even though production yield has been increasing for major saffron producers over the years, Spain has been often been the leader. Fig. 21.12 shows yields in Iran, Greece, and Spain. Saffron yield in Spain increased from 10 to 14 kg ha21 from 2005 to 2016, a 42% increase in growth. Greece is in second place with an average yield of about 6 kg/ha. Among the three countries, Iran has the lowest yield with an average of 3.13 kg ha21. The large reduction in yield for 2008, as compared to that in the previous year, was due to frostbite weather in the two major saffron-producing provinces of South and Razavi Khorasan in 2007 (Iranian Ministry of Agriculture, 2017).

21.5

Production function in economics

The production function is the relationship between physical output of a production process and physical inputs or production factors. The main purpose of estimating a production function is to determine whether goods and services are produced in the most efficient and economical way. Indeed, the production function is the output obtained from the most combination of inputs.

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SECTION | V Economy and trade of saffron

The production function is practically applied by valuing the physical outputs and inputs using their prices. By comparing the economic value of the last unit of production with the cost incurred to produce it, we can determine if the use of more production inputs leads to more economic benefits. If the economic value obtained from the last unit of production is higher than the production cost, it is economically beneficial for the producer to use more units of the inputs. Alternatively, if the production cost of the last unit is higher than the benefits obtained from producing it, economic benefit is increased by reducing input use. Therefore, the most economic benefit is obtained at the point where the economic benefit of the last unit of production equals its production cost.

21.5.1 Production function of saffron In saffron cultivation, the main production inputs are land, labor, irrigated water, manure, chemical fertilizer, saffron corm, agricultural machinery, and herbicide. Studies on the production function for saffron cultivation normally use a Cobb
Douglas production function: Yi 5 A Xij β j 1 ei

(21.1)

where Yi is the yield of saffron for the ith farmer, Xij is the jth input used by the ith farmer, β j is coefficients for inputs estimated by the model, and ei is the error term. To simplify the model estimation, it can be linearized as LnYi 5 LnA 1 β j Ln Xij 1 ei

(21.2)

The results of estimating a Cobb
Douglas function of saffron cultivation show that the economic importance of production inputs varies across various cultivation regions (Golkaran-Moghadam, 2014; Jalali et al., 2015, 2016; Khanali et al., 2016). In the Torbat-e-Heydarieh county, expansion of the farming area (Golkaran-Moghadam, 2014; Jalali et al., 2015, 2016), an increase in labor per hectare (Golkaran-Moghadam, 2014; Jalali et al., 2016), corm density (Golkaran-Moghadam, 2014), manure use (Golkaran-Moghadam, 2014), chemical fertilizer use (Golkaran-Moghadam, 2014), agricultural machinery use (Jalali et al., 2016), and water irrigation use (Jalali et al., 2016) increase economic benefits obtained from saffron cultivation, while an increase in herbicide use per hectare will decrease the economic benefit (Golkaran-Moghadam, 2014). In the two counties of Ghaen and Gonabad, four inputs—land, labor, chemical fertilizers, and irrigated water—increase economic benefits, but the use of more herbicides and manures are expected to decrease economic benefits (Golkaran-Moghadam, 2014; Khanali et al., 2016). Putting all these results together, while labor and irrigated water inputs had a significantly positive impact on the economic benefits obtained from saffron cultivation (Bakhtiari et al., 2015), herbicides had a negative impact in all three regions. The negative impact is due to the overuse of herbicides due to the lack knowledge about their use.

21.6

Economic productivity of saffron

Productivity is an economic tool used to compare some alternative economic activities in terms of their obtained benefits. Productivity is a measure of output per unit of input. Table 21.2 shows the inputs used for saffron cultivation in Torbat-e-Heydarieh, the major area for saffron production in Iran. Data in the table are based on a questionnaire TABLE 21.2 The amount of inputs used to produce 1 kg of saffron. Inputs

Amount

Inputs

Nitrogen fertilizer (kg)

20.2

Gasoline (kg)

Amount 18.99 21

Phosphorus fertilizer (kg)

12.84

Electricity (kW h )

18.84

Potash fertilizer (kg)

14.07

work force (people)

21

Manure (tons)

4.76

Irrigation (h)

3.46

Micronutrient (L)

0.26

Machinery (h)

2.55

Herbicide (kg)

0.09

Corn (tons)

0.18

Source: Data from Nezamoleslami, A., 2018. The Comparative Advantage of Saffron Production in Khorasan Razavi Province with Regard to Greenhouse Gas Emissions. Case Study: Torbat-e-Heydarieh Region (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

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345

completed by 57 farmers who cultivated saffron in 2017. Table 21.2 shows the average of each input used by the farmers to produce 1 kg of saffron. The main inputs of saffron cultivation are water, land, and labor, so we analyze the productivity of these inputs in the rest of this chapter.

21.6.1 Water productivity of saffron One of the most important factors in determining suitable agricultural systems for arid and semiarid regions (e.g., water-scarce countries) is water productivity or the value of production per a unit of water used. Many sources (Amirteymouri and Khaliliyan, 2008; Khavarinezhad, 2014; Shamsabadi et al., 2016) define water productivity as the ratio of product yield to the amount of water used in production. With this aim, studies on various agricultural products measured the amount of production per cubic meter of water used. For instance, 1 m3 of water can generate 1 g of saffron, 1 kg of wheat, 6 kg of potatoes, and 6.5 kg of sugar beets (Koocheki et al., 2017), as reported by the Iranian Students News Agency (ISNA, 2017). According to Zwart and Bastiaanssen (2004), a cubic meter of water can be used to produce 0.99 kg of wheat, 1.9 kg of rice, 0.65 kg of cotton, and 1.80 kg of corn. While this information is helpful for farmers and policymakers to know the amount of water required for planting each product, from an economic viewpoint it is necessary to determine the monetary value of product per 1 m3 of water used. This allows an economic comparison among different types of products. It is instructive to compare the income generated per cubic meter of water in its various uses. Each cubic meter of water used for saffron production generates 9 times more income than wheat, 10 times more than sugar beet, 6 times more than potato and 2.5 times more than apple (Sadeghi, 2012). Another study (Tabatabaei and Shahidi, 2017) analyzed a scenario of eliminating cultivation of wheat, barley, and alfalfa, and replacing these crops with saffron. This study shows that reallocating water to saffron from barley increased net income by 5.8 times. This study also showed that removing wheat, barley, and alfalfa production and allocating the water consumed to saffron could increase the area under saffron cultivation by 6.8 ha (reaching 46.8 ha) based on traditional irrigation (e.g., 4500
5000 m3 of water for a hectare of saffron). Drip irrigation would save 1000 m3 of water per hectare. In a summary, the survey of existing studies and reports shows that low water requirements, high tolerance to drought/water shortage, soil fertility, and high levels of economic productivity of water in saffron cultivation make saffron a suitable product for arid and low water areas.

21.6.2 Labor productivity of saffron The economic productivity of labor is the amount of income generated by a unit of labor. Labor productivity shows how much a laborer produces in output value. Although saffron production is very labor-intensive, especially the planting and harvesting stages, the high level of income produced from this crop yields high labor productivity in comparison with other crops. Labor productivity for saffron is 2.4 times more than wheat, 3.6 times more than barley, 4.6 times more than cotton, and almost 10 times more than a vine crop (Monazam-Esmaeilpour and Kordovani, 2010). As a result, the production of this product (in comparison with other agricultural products) creates high income for employees in this sector. The high income generated by saffron can have even more positive effects on the status of poor workers. Increased wages are expected to positively affect school enrollment and birth rates (Becker, 1992). Nevertheless, saffron is not economically suitable for regions with high labor wages. For instance, in New Zealand high labor costs limit saffron production even though its yield is high (up to 24.3 kg ha21; more than three times the yield of traditional producing countries) (McGimpsey et al., 1997).

21.6.3 Land productivity of saffron Saffron cultivation is also high in terms of land productivity. Saffron yields differ by country due to climatic conditions and cultivation methods. But studies show that the value-added generated by saffron cultivation is several times higher than wheat, beets, and other crops (Kouzegaran et al., 2013; Tabatabaei and Shahidi, 2017). The results of the study by Monazam-Esmaeilpour and Kordovani (2010) show that the net income of 1 ha of saffron is 16 times more than wheat, 24 times more than barley, 19 times more than cotton, 31 times more than wheat, 7 times more than grapes, and 45 times more than pistachio.

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SECTION | V Economy and trade of saffron

Economic performance of saffron

We use some economic indicators to analyze the economic performance of saffron production.

21.7.1 Return on investment Return on investment (ROI), sometimes known as rate of return, is the percentage change in the value of an investment over a certain period of time. The following formula is used to measure it: ROI 5

Vf 2 Vi  100 Vi

(21.3)

where Vf is the value of the investment at the end period of time and Vi is the initial value of investment. The average ROI for saffron cultivated in Torbat-e-Heydarieh, the major saffron region in Iran, is about 500%. In other words, a saffron field, which lasts 5
7 years, generates income that is five times more than its establishment costs.

21.7.2 Ratio of return to cost The ratio of return to cost (RRC) is another economic indicator used to evaluate the efficiency of an economic activity and investment. This ratio is calculated using annual income from an investment divided by the cost of the investment. The result is either a ratio or percentage. In Torbat-e-Heydarieh, the average RRC for saffron production is 2.1, while the ratio is 1.2, 1.12, and 1.41 for wheat, barley, and alfalfa production, respectively. The RCC for sugar beets is estimated between 1.02 and 1.56, depending on the production scale (Gholami-Ghajlou et al., 2014). Saffron’s RRC is about two times more than this ratio for the mentioned products (Salehi and Karimiyan, 2017).

21.8

Efficiency

In evaluating the ecological performance of a human activity, we may take various perspectives. One of the most common notions used is “efficiency,” which is the distance between what is actually performed and what can be ideally achieved (Sherman and Zhu, 2006). However, the challenging point on the concept of efficiency is that experts and researchers in different areas of research conceptualize “ideal achievement” differently. For an ecological scientist, the achievements are considered based on energy produced during a plant’s growing period (Sherman and Zhu, 2006). However, the produced energy can be measured in different forms such as assimilation, known as assimilation efficiency (Jahan et al., 2012), and consumer production, known as production efficiency (Khavarinezhad, 2014). From an economic perspective, “ideal achievement” is generally defined by the economic benefits obtained from the production process (Sherman and Zhu, 2006). Nevertheless, the economic benefit can be analyzed from different perspectives such as input combination (Sherman and Zhu, 2006) or production scale (Sherman and Zhu, 2006). From a social point of view, “ideal achievement” is usually in terms of resource distribution. Indeed, maximum efficiency in a society is achieved when making one person better off requires someone else to be worse off (Barr, 2004). Nevertheless, the value associated with efficiency, in any of its forms, is between 0 and 1. In many studies of ecological performance (Abdshahi et al., 2013; Taki et al., 2011), energy efficiency is used to assess how energy moves in the production process. The following sections describe energy efficiency in saffron producing.

21.8.1 Energy use efficiency Energy efficiency is how energy is consumed as the product moves from raw material to final product during the production process. Indeed, energy efficiency is energy delivered by the product compared to the energy consumed to produce it. The following formula measures energy efficiency: Energy use efficiency 5

Energy in the output ðMJ ha21 Þ Total energy in the inputs ðMJ ha21 Þ

(21.4)

Economic analysis of saffron production Chapter | 21

347

To calculate energy use efficiency, four stages are followed: Stage 1: Specify the inputs used for the production cycle (which lasts 5
7 years for saffron) per hectare. For conventionally produced saffron, the main production inputs are fossil fuels used for machinery, fuels used to generate electricity, different types of fertilizers (such as farmyard manure), other chemicals such as herbicides and pesticides, human labor, irrigation water, and corms (Bakhtiari et al., 2015; Khanali et al., 2016; Khoramdel et al., 2016; Yaghoubi et al., 2015). Stage 2: Convert the physical quantities of the inputs used during the production cycle per hectare into energy values. To quantify the energy values, most studies (Bakhtiari et al., 2015; Elhami et al., 2016; Ghorbani-Birgani and Dibaei, 2010; Heidari-Soltanabadi, 2014; Khanali et al., 2016) use energy coefficients drawn from the literature. For each input an energy coefficient indicates the energy used to produce one unit of the input from the beginning of the production process to the end user (Mousavi-Avval et al., 2011a,b). One has to consider the technique used to produce the inputs applied in the production process (Mousavi-Avval et al., 2011a,b). Table 21.3 presents energy coefficients for the main inputs required for saffron production under conventional methods. Stage 3: Specify the production outputs. In this context, various levels of outputs are distinguished. However, since the efficiency term refers to the human benefits gained, studies (Karunarathna and Wilson, 2017; Maia et al., 2016) only consider parts of the plant that benefit humans as “economic output.” In saffron, the economic output is stigma and corms, with leaves and flowers as byproducts (Bakhtiari et al., 2015). Stage 4: Convert the physical quantities of the outputs produced over the production cycle per hectare into energy values. Studies have used two techniques for the quantification of output energy. Similar to the input energy quantification, some studies (Khanali et al., 2016; Mehrabadi-Basharabadi and Pourmoghadam, 2012; Mirhosein et al., 2005; Mousavi-Avval et al., 2011a,b) use energy coefficients calculated by other studies (FAO, 2000). Other studies (Bakhtiari et al., 2015; Ghaderpour and Rafiei, 2016; Ziyaei et al., 2013) use laboratory methods to calculate energy output of agricultural products. The efficiency is the ratio between the caloric useful products and the total energy used to produce inputs (EsmaeiliDastjerdipour and Mehrabi-Basharabadi, 2011). This clarifies whether the production process is a net producer or consumer of energy (Khanali et al., 2016).

TABLE 21.3 Energy coefficients of different inputs and outputs used in agricultural production. Energy coefficients (MJ unit21)

Inputs

Units

1. Human labor

h

2. Machinery

h

62.7

Mobtaker et al. (2010), Nabavi-Pelesaraei et al. (2014)

3. Diesel fuel

L

56.31

Mobtaker et al. (2010)

4. Electricity

kWh

11.93

Mobtaker et al. (2010), Mohammadi et al. (2014)

5. Chemical fertilizers

kg

a. Nitrogen (N)

66.14

Esengun et al. (2007), Mousavi-Aval et al. (2011)

b. Phosphate (P2O5)

12.44

Esengun et al. (2007), Mousavi-Aval et al. (2011)

11.15

Esengun et al. (2007), Mousavi-Aval et al. (2011)

1.96

c. Potassium (K2O) 3

Reference Beheshti-Tabar et al. (2010), Mohammadi et al. (2008)

6. Water for irrigation

m

0.63

Erdal et al. (2007)

7. Farmyard manure (FYM)

kg

0.3

Demircan et al. (2006)

8. Chemicals

kg

a. Pesticides

199

Ozkan et al. (2004)

b. Herbicides

238

Helsel (1992)

c. Fungicide

92

Ozkan et al. (2004)

Source: Data from Bakhtiari, A.A., Hematian, A., Sharifi, A., 2015. Energy analyses and greenhouse gas emissions assessment for saffron production cycle. Environ. Sci. Pollut. Res. 22 (20), 16184
16201.

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21.8.1.1 Energy use efficiency of saffron cultivation All published studies on the energy efficiency of saffron cultivation have been conducted in Iran; we did not find studies conducted in other countries. Having reviewed these studies, the following points can be noted: 1. The first published study on the energy efficiency of saffron cultivation was conducted by Jami-Al-Ahmadi et al. (2010) for the Southern Khorasan province. In this study, energy efficiency was calculated at 0.006. The two other studies reported energy efficiency for saffron of 0.0026 for the provinces of Khorasan Razavi and Southern Khorasan (Bakhtiari et al., 2015) and 0.0044 for Southern Khorasan province (Khanali et al., 2016). The three studies confirmed low energy efficiency for saffron cultivation compared to other agricultural products. The main reason for the low energy efficiency of saffron is that only a small part of the plant (the stigma) is an economic output. The ratio of usable yield or economic yield to the total amount of the above ground biomass is 0.5% for saffron, while it is usually between 30% and 60% for most agricultural products (Kafi, 2006). 2. Farm-level energy consumption in Iran varies tremendously. This indicates that the production process varies widely from farm to farm. 3. Fertilizers, in particular N-based fertilizers, constitute most of the energy consumption. As a result, managing fertilizer consumption in saffron should be given priority. Other studies (Mohammadi et al., 2014; Mousavi-Avval et al., 2011a,b; Sefeedpari et al., 2014) also indicate that chemical fertilizers, in particular N-based fertilizers, are excessively employed in Iran. 4. Land preparation consumes most of the energy input during the saffron production cycle. This is because fuel used in machines and most fertilization takes place when the soil is being prepared for planting.

21.8.1.2 Economic efficiency Economic efficiency measures whether resources are allocated to produce the desired outputs in the most economical way. Economic efficiency is obtained when a product is produced at the lowest possible cost. Therefore, at the point of economic efficiency, any change made to improve a part of the production process would harm another part. When economic efficiency is achieved, nothing can be improved without something else being hurt (Farrell, 1957). Economic values of consumed inputs and produced outputs are the main determinants of economic efficiency. Estimating the degree of a firm’s efficiency shows how the firm can improve economically by either reducing its inputs without changing its production or expanding production without changing its inputs. Economic efficiency compares firm operations that produce under a common technology. Efficiency falls in a range between 0 and 1. A value of 1 indicates the firm is an efficient unit and a value less than one denotes the firm is inefficient. An inefficient unit can improve in two ways: 1. By reducing its input consumption while output remains constant 2. By expanding its output while inputs remain constant The efficiency comparison can be conducted in two forms—input-based efficiency and output-based efficiency (Bogetoft and Otto, 2011). The input-based efficiency under a certain technology “T” is:    E 5 min E . 0ðEx; yÞAT (21.5) where E, x, and y are the efficiency, the inputs, and the outputs, respectively. This input-based efficiency presents the maximum reduction of inputs that allows the firm to continue producing a given output. On the other hand, the outputbased efficiency under a certain technology “T” is:    (21.6) F 5 max F . 0ðx; FyÞAT where F, x, and Fy are the efficiency, the inputs, and the outputs, respectively. This output-based efficiency denotes the maximum increase in output that is feasible consuming the given inputs. 21.8.1.2.1

Economic efficiency of saffron cultivation

Studies on the economic efficiency of saffron cultivation have been mainly conducted in Iran. The main findings are: 1. Saffron is mainly cultivated in the two neighboring provinces of Khorasan Razavi and Southern Khorasan located in northeastern Iran. The two counties of Torbat-e-Heydarieh and Gonabad from Khorasan Razavi and the county of Ghaen from Southern Khorasan are the major regions of saffron production in Iran. Findings from different studies

Economic analysis of saffron production Chapter | 21

2.

3.

4. 5.

349

on economic efficiency for saffron production in those regions show average economic efficiency between 80% and 90%, indicating that production can increase more than 10% without increasing inputs (Golkaran-Moghadam, 2014; Kavand et al., 2014). More specifically, among these three regions, Torbat-e-Heydarieh, with an average of economic efficiency of 86%, is the most efficient area in producing saffron, and Ghaen, with an economic efficiency of 63%, is the lowest one (Golkaran-Moghadam, 2014). Jalali et al. (2016) found that the benefit efficiency of saffron (61.4%) was significantly less than the economic efficiency (86%) in Torbat-e-Heydarieh. This indicates that the net benefit from saffron production can be raised on average by 38.6% without any change in the production scale. The results of this study revealed that saffron producers were not able to make the most efficient decisions about inputs due to fluctuating input prices and input price uncertainty. In the Kozani area of western Macedonia, the main Greek area for saffron cultivation, the economic efficiency of saffron was estimated at 63% under constant returns to scale indicating that the production inputs can decrease by 37% while leaving production unchanged (Melfou et al., 2015). Firm-level economic efficiencies varied significantly among firms due to management and input use (GolkaranMoghadam, 2014; Kavand et al., 2014). Some socioeconomic characteristics, such as age, education, number of farms, and off-farm income sources, were the main determinants of economic performance for saffron farms. Two farmer characteristics, age and education, have positive effects on farm efficiency of saffron production. Older and more educated farmers have higher farm efficiency, indicating better management of resources.

21.9

Economic comparative advantage

21.9.1 Revealed comparative advantage Revealed comparative advantage (RCA), also known as the Balassa index, is a trade index used to analyze the comparative advantage or disadvantage of a certain country in producing and exporting a product or service. The Balassa index is based on Ricardo’s theory that countries allocate their resources to produce products in which they have comparative advantages. Therefore, a product with a larger share in a country’s exports has a comparative advantage for that country. The RCA compares a country’s export pattern relative to the world. The RCA of a country for a given product is:   PXmij Xij  RCAij 5  Pj51 (21.7) n Xij i51 Pn Pm j51

j51

Xij

where X is exports, i denotes the country, j denotes the product, m is a set of countries, and n is a set of commodities. Comparative advantage is revealed when RCA .1. The Balassa’s index has been used in many studies (Amirnezhad and Alipour, 2013; Mehrabadi-Basharabadi and Pourmoghadam, 2012) to calculate the comparative advantages of a country’s products.

21.9.2 Policy analysis matrix The theory of comparative advantage developed by David Ricardo (Ruffin, 2002) indicates that a country will reach higher economic growth by promoting industries in which it has the greatest comparative advantage. Comparative advantage is present if a country produces goods and services that have a low opportunity cost. It shows if the benefits of producing a good or service outweigh the costs. The comparative advantage of a particular product can be analyzed using the PAM in which two sets of prices, private and social, are considered. Private prices are determined by the market; indeed, traders face private prices directly in the market. Social prices are opportunity costs for outputs and inputs, so they reflect externalities from production. International prices are normally thought of as social prices (unless there are externalities), but domestic resource values are determined in local markets. A PAM, shown in Table 21.4, can be used to analyze economic comparative advantage, efficiency of using production inputs, and differences between social and private costs.

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TABLE 21.4 Policy analysis matrix. Values

Revenue

Cost

Profit

Tradable inputs

Domestic inputs

At private prices

A

B

C

Da

At social prices

E

F

G

Hb

Differences

Ic

Jd

Ke

Lf

Private profits, D 5 A 2 (B 1 C). Social profits, H 5 E 2 (F 1 G). Output transfers, I 5 A
E. d Input transfers; J 5 B
F. e Factor transfers, K 5 C
G. f Net policy transfers, L 5 D
H. Source: Data from Monke, E.A., Pearson, S.R., Akrasanee, N., 1976. Comparative advantage, government policies, and international trade in rice. Food Res. Inst. 15 (2), 1
27. a

b c

TABLE 21.5 Inputs consumed and output produced during 1 year (per hectare). Input

Average

Nitrogen fertilizer (kg)

Input

Average

Micronutrient (L)

Input

Average

Corm (tons)/seeder (h)

First year

115.78

First year

1.82

Corm (first year)

7.55

Second and years after

119.29

Second and years after

0.85

Seeder (first year)

11.54

Phosphorus fertilizer (kg)

Herbicide (L)

Tillage (h)

First year

78.07

First year

0.72

Second and years after

79.82

Second and years after

0.71

Potash fertilizer (kg)

Labor (person)

First year

83.33

First year (person)

Second and years after

87.71

Second and years after

Manure (tons) First year

29.01

Second and years after

13.23

Second and years after

14.85

Irrigation (h) 77

Each year

5.94

177

Fossil fuel (L)

Yield (kg)

Second and years after 199.38

Yearly except the 1th year

7.21

Source: Data from Nezamoleslami, A., 2018. The Comparative Advantage of Saffron Production in Khorasan Razavi Province with Regard to Greenhouse Gas Emissions. Case Study: Torbat-e-Heydarieh Region (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

Yields, inputs, output, and market and social prices of inputs and outputs are the main information required to calculate a PAM. Table 21.5 presents the average amount of inputs consumed for 1 ha of saffron located in theTorbat-eHeydarieh region (Nezamoleslami, 2018). The determination of social prices is the most important part of PAM calculations. To estimate social costs, the study (Nezamoleslami, 2018) divided inputs into tradables, such as machinery, herbicide, fertilizer, and corm that can be exported or imported, and nontradables, such as labor, land, and water that cannot be exported or imported. For tradable inputs, cost, insurance, and freight (CIF) prices are used as social costs, and an opportunity cost (the highest market price) is used for nontradable inputs. The fob (free on board) price of saffron is used for social income (Table 21.6). Fig. 21.13 shows the market and social (shadow) prices for each input used to produce saffron. Table 21.7 shows a PAM estimated for saffron production in Torbat-e-Heydarieh in 2017. During a 7-year cycle of saffron cultivation, the income earned for 1 ha based on shadow prices is 217,500,000, which is 21,750,000 rials more than the market income for the same amount of saffron. This implies that there is an implicit 10% tax on saffron production because market prices in Iran are lower than international prices for saffron.

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351

TABLE 21.6 Income, the cost of tradable and nontradable inputs at shadow, and market price for a hectare of saffron farm (in dollars). Items

Unit

Market

Shadow

Income

1 kg of saffron

375

416.67

Tradable inputs Rental of machinery

Used during a year

4.02

40.43

Nitrogen fertilizer

Used during a year

8.10

10.95

Potash fertilizer

Used during a year

0.01

17.04

Phosphorus fertilizer

Used during a year

11.57

13.93

Micronutrient

Used during a year

8.65

10.25

Herbicide

Used during a year

0.79

8.97

Manure

Used during a year

141.59

141.59

Saffron corm

Used in the first year

262.35

262.35

Labor

Used during a year

293.8

536.46

Rental of land

Used during a year

224.63

356.73

Irrigation

Used during a year

312.35

490.87

Nontradable inputs

Source: Data from Nezamoleslami, A., 2018. The Comparative Advantage of Saffron Production in Khorasan Razavi Province with Regard to Greenhouse Gas Emissions. Case Study: Torbat-e-Heydarieh Region (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

FIGURE 21.13 Input prices at market and social levels (dollars per hectare). Data from Nezamoleslami, A., 2018. The Comparative Advantage of Saffron Production in Khorasan Razavi Province with Regard to Greenhouse Gas Emissions. Case Study: TTorbat-e-Heydarieh Region (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

For nontradable inputs, the difference between market prices and shadow prices is Rs 40,200,000, indicating that saffron producers receive an indirect subsidy for nontradable inputs. Thus government programs and policies reduce private production costs by 68%. The value of tradable inputs calculated based on market prices is Rs 426,626, which is less than the value based on social prices. This indicates that saffron producers are receiving a subsidy of about 2% on tradable inputs. According to the profit calculations, producing saffron is profitable at both market prices (Rs 81,449,439) and shadow prices (Rs 62,572,813). Since the profit at market prices is greater than the profit at shadow prices, there is a net subsidy to saffron producers. The PAM provides three indicators of comparative advantage: Nominal Protection Coefficient (NPC), Effective Protection Coefficient (EPC), and Domestic Resource Cost (DRC). In the next section, we present the comparative advantage indices obtained by our study and the results from other studies.

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TABLE 21.7 Policy analysis matrix for saffron crop, rials per hectare, for a 7-year period, Torbat-e-Heydarieh, 2017. Value

Income

Costs Tradeable inputs

Profit Nontradable inputs

At market price

195,750,000

29,943,544

84,357,018

81,449,439

At shadow prices

217,500,000

30,370,170

124,557,018

62,572,813

2 21,750,000

2 426,626

2 40,200,000

18,876,626

Deviations

Source: Data from Nezamoleslami, A., 2018. The Comparative Advantage of Saffron Production in Khorasan Razavi Province with Regard to Greenhouse Gas Emissions. Case Study: Torbat-e-Heydarieh Region (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

21.9.3 Nominal protection coefficient The NPC is the ratio of domestic price to social price. The domestic price can be a farm-gate price, while the social price is the international price adjusted for transportation, marketing, and processing costs. This indicator measures external policies including tariffs, subsidies, or other distortions causing a divergence between the two prices. For a given good, an NPC with a value higher than one shows that national policies push the domestic price higher than the international price. On the other hand, an NPC less than one indicates that national policies reduce the market price compared to its international price. The indicator can be calculated for both inputs (NPCI) and outputs (NPCO). The NPCI addresses how much the domestic prices of inputs differ from their social prices. If the domestic input cost is higher than the international price (NPCI . 1), there is net taxation. If the domestic input cost is lower than the international price (NPCI , 1), there is net subsidization. The NPCO can be analyzed in the same way; an NPCO . 1 implies subsidization (private price above social price for output) and an NPCO , 1 implies taxation. In our study, the NPCI is estimated at 0.99. The index was estimated at 0.53 by (Najarzadeh et al., 2010). Both results indicate that farmers receive subsidies for their production factors. The NPCO was estimated at 0.9, and was 0.62 from Aghai and Rezagholizadeh (2011), indicating that the international price of saffron is higher than the domestic market price, and therefore saffron production is taxed indirectly.

21.9.4 Effective protection coefficient The EPC is a relative comparison of value added in private cost (A
B in Table 21.5) to value added in social cost (E
F in Table 21.5). EPC 5

A2B E2F

(21.8)

The EPC shows the impact of protective policies on input and output prices, and reveals the level of protection for the value added. If the EPC is higher than one (EPC . 1), the value added at market price is higher than it would be at social cost, so the domestic market is protected. If the EPC , 1, the domestic market is taxed, so producers are discouraged. In our study, we estimated EPC at 0.89, while Najarzadeh et al. (2010) estimated saffron’s EPC at 0.63. Both results show that saffron production is not protected, but taxed, so policies are discouraging producers.

21.9.5 The domestic resource cost The DRC is the most useful and common indicator to analyze economic comparative advantage for agricultural commodities. The indicator is computed using the ratio of nontradable factors used to produce the output per unit of tradable value added: DRC 5

G E2F

(21.9)

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353

The DRC shows the shadow value of nontradable factors per unit of tradable value added. The rationale for using DRC is that there are commonly many domestic distortions in input markets. If DRC . 1, the use of domestic resources is not socially profitable. If DRC , 1, domestic resources are socially profitable. The DRC of producing saffron on farms located in the Khorasan Razavi and Southern Khorasan is estimated at more than 0.8 (Aghai and Rezagholizadeh, 2011; Karbasi and Rastegaripour, 2014; Najarzadeh et al., 2010), indicating that generating one unit of income from the international market costs about 0.8 units. In Table 21.5 the DRC of producing saffron in Torbat-e-Heydarieh is estimated at 0.67. This implies that Iran has a comparative advantage in producing saffron.

21.9.6 Based on unit costs The UC is an alternative to DCR in which all costs are considered and the classification error of the DRC is avoided (Masters and Winter-Nelson, 1995). The indicator is: UC 5

F1G E

(21.10)

A UC , 1 shows that the production process generates a surplus over costs and a UC . 1 indicates that the production process is not profitable. The UC for the Khorasan Razavi and Southern Khorasan were estimated at 0.88 (Aghai and Rezagholizadeh, 2011; Najarzadeh et al., 2010). For the area we studied (Torbat-e-Heydarieh presented in the Table 21.5), the UC was estimated at 0.71. Both results imply that saffron production is profitable at current social prices.

21.10 Some economic advantages of saffron production Small-scale production: An important economic advantage of producing saffron is the possibility of planting on a small scale. The crop can be produced easily under land constraints. In this regard, farmers can cultivate saffron in spaces among trees for more productive use of water and land. Employment opportunities: Saffron is a labor-intensive product and is dependent on manual work, like handicrafts. The saffron harvest is similar to making handicrafts in that it uses only labor to extract the stigma from the flower. In general, saffron production requires 200
205 man days per hectare. Wheat, barley, and vine crops require 32, 69, and 58 man days per hectare, respectively (Monazam-Esmaeilpour and Kordovani, 2010). Of the total labor required for saffron production, 10% is needed for the planting stage, 25% for the cropping stage, and 65% for the harvesting stage. Saffron production provides a great wage/work opportunity for females. The employment opportunities in saffron cultivation have moved areas that have great potential for other crops toward production of saffron. There are also many job opportunities in the agribusiness sector beyond the harvesting stage to the final sale and even export for saffron. Product processing and the production of quality packaging are among the most important issues that must be addressed if the agribusiness sector is to expand for saffron. Considering the importance of saffron in the world market, and the employment needs in rural Iran, improved saffron production and marketing can generate many employment opportunities. Nonadvanced equipment and facilities: Saffron cultivation does not require sophisticated machines and equipment. This makes it possible for most farmers, even those with low income and little financial capital, to produce saffron. Since saffron production generates more income compared to other agricultural products with limited investment, the expansion of saffron production has the potential to reduce poverty. However, when the cultivated area expands beyond the market demand, competition may change these results, so producers must keep in mind that large increases in production may lower price.

21.11 Conclusion Saffron is a unique crop with a very high economic value compared with the other agricultural products. Its price is mainly affected by climatic conditions, in particular drought spells, and global economic factors such as economic crisis. Political and environmental factors have only a small influence on the saffron trade. However, the usable yield (e.g., the part of product that is used for human purposes, mainly stigma) in terms of energy or amount of production is very low. Due to the high price, productivity of the inputs used in production is very high. Saffron has a comparative advantage in regions with water shortage, nonadvanced agriculture, and low labor wage.

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133.

Chapter 22

Saffron marketing: challenges and opportunities Hosein Mohammadi1 and Michael Reed2 1

Department of Agricultural Economics, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran, 2Department of Agricultural

Economics, College of Agriculture, Food and Environment, Kentucky University, Lexington, KY, United States

Chapter Outline 22.1 Introduction 22.2 Problems of exporting and marketing of saffron 22.3 Marketing concepts in the saffron industry

22.1

357 357 360

22.4 Marketing management tasks for saffron marketing 22.5 Conclusion References

362 364 365

Introduction

Saffron is one of the most expensive agricultural and medicinal products in the world. Specific features of saffron, such as family labor requirements, low water requirements, ability to grow in clay and sandy soils, minimal machinery needs, and the potential acceptable income for farmers have led to an increase in the cultivation and production of saffron continuously in countries such as Iran. Iran, Greece, Morocco, Spain, and Italy are the largest saffron producers in the world, and beginning in 2015, the neighboring countries of Iran such as Afghanistan entered this market. Saffron is a strategic product in the southern Khorasan and Khorasan Razavi provinces in Iran, as it can help create seasonal and permanent employment for people, prevent migration, generate income, and expand nonoil exports. Iran accounts for more than 90% of the world’s production of saffron and 80% of Iran’s production of saffron comes from these two provinces. Iran exports more than 80% of its saffron to the United Arab Emirates (UAE), Spain, France, and Italy. The mentioned countries reexport Iranian saffron to targeted countries after making modest changes such as processing, but capturing huge value-add. Torkamani (2000) reported that production, processing, and marketing problems in Iran have encouraged raw saffron to be exported without important value-adding features. Iran tends to export saffron in bulk, minimizing its value-add. This phenomenon is characteristic of many Iranian raw materials, such as oil and gas, but also basic agricultural products like medicinal herbs. Raw materials export reduce profits as well as decreasing the competitiveness of the country in the world markets. Countries like the UAE and Spain, by importing bulk Iranian saffron and then packing, grading, and producing related products, achieve more value-add from Iranian saffron than the originating country.

22.2

Problems of exporting and marketing of saffron

Hashempour (2009), Hosseini and Aho-Ghalandari (2007), Pezeshkirad and Feali (2010), and Torkamani (2000) noted several issues facing the exporting and marketing of saffron. The most important factors include: 1. Noncompliance with hygiene standards as the product is harvested, processed, and packaged. Most saffron consumers are looking for a high-quality, safe, hygienic product, so it is important to observe hygiene throughout the various stages of saffron production and processing. Saffron is used as a food ingredient, natural color agent, herbal Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00022-8 © 2020 Elsevier Inc. All rights reserved.

357

358

2. 3.

4.

5.

6.

7. 8.

9.

SECTION | V Economy and trade of saffron

medicine, and many other applications in various industries. Therefore, it is essential to observe health and safety protocols for this product that are compliant with international standards. Bulk exports of saffron. In order to increase exports of high value-added saffron products, saffron-related industries must compete with other countries that buy and reexport the saffron with their own brands and packaging. The saffron industry should pay attention to customer wants and needs in target markets. Consumers buy saffron for different uses, including pharmaceutical, health, and food industries. The saffron industry in producing countries might do a better job of meeting customer needs if more of the product was processed in their countries. Saffron producers and marketers could produce and handle the product in a way that generates and preserves its most desirable characteristics, which can create more value. Marketing costs can change consumer expenditure, so efforts to reduce marketing and production costs will increase consumer welfare and producer revenues. Hosseini and Aho-Ghalandari (2007) reported that the marketing margins for saffron are directly related to its retail price and that marketing costs such as wage and transport costs increase retail prices. The lack of mandatory standards for production and processing of saffron is a problem because there is too much variation in quality. The lack of well-equipped laboratories and specialized workers to enforce mandatory standards for production and procession of saffron are a requisite for producing high-quality saffron products. The presence of educated and knowledgeable people in the saffron trade can play a significant role in improving markets and exports for this sector. Most of the companies that are active in the saffron industry use their own experience without using scientific marketing and trade principles. Therefore, their results are disappointing and countries export raw product instead of highly processed items, reducing profits significantly. The lack of enough knowledge about potential foreign market opportunities for saffron and its products is another problem in the marketing of saffron. Saffron is widely used in the food, sanitary, and cosmetics industries as well as in the dye and chemical industry, in addition to others, but there is lack of knowledge and technology in some producing countries to turn raw saffron into these high-valued subproducts. There is no prestigious brand for saffron in some producing countries. This makes it difficult to establish further processing and leads to difficulties in e-commerce.

Mosavi et al. (2013) argued that Iran has been the largest producer of raw saffron in the world for the past two decades, but there are doubts about its ability to maintain this position in the future. Due to its special climate, Iran is suitable for saffron cultivation in its arid and semiarid regions, like South Khorasan, Khorasan Razavi, Fars, Kerman, and Yazd provinces, despite being water scarce. There are some problems with saffron production and processing methods because it is still performed in a very traditional structure and manner, but most of the problems involve exportation of this valuable herb. Lack of attention to international standards, antiquated distribution systems, and lack of a central organization to monitor and production, advertising, and exporting of saffron create several challenges. Currently, a handful of countries distribute this product to global markets. These countries mainly purchase saffron at a low price from producing countries, provide some low-level processing, gain a high value-add, and reexport it to global markets under their brand names. Some producing countries have failed to capitalize on their role as exclusive producer of raw saffron and have suffered as a result. One factor that prevents these countries from gaining more benefit from saffron (and allows other countries to gain), is its inappropriate packaging and bulk export of the product to global markets. Spain and UAE mainly purchase saffron in bulk, repack it, and distribute it to the global markets under their brand names, enjoying a rich margin (Aghdaie and Roshan, 2015). Pezeshkirad and Feali (2010) investigated the challenges of saffron marketing, processing, and exporting, and concluded that the main challenges include: G G G G G G G G G

Lack of suitable packaging industries Noncompliance with international standards Lack of reliable and global brands for saffron Traditional production and processing methods Inadequate exporters training Poor promotional practices in global markets Lack of effective marketing organizations Weakness earning power of saffron growers High presence of unnecessary brokers in the saffron market

Saffron marketing: challenges and opportunities Chapter | 22

G G G G G G G

359

Lack of information on world markets Fluctuations in saffron prices Government policies and regulations Lack of research funding and failure to conduct research on the properties and subproducts of saffron Incidence of fraud Entry of new competitors such as Afghanistan and China Weakness in electronic commerce

Furthermore, they suggest that the most important strategies to improve the marketing and exporting of saffron include developing: 1. Government policies to provide loans and support producers and processors to create standardization of packaging for saffron exports 2. Commercial and business training courses for exporters 3. Research specializing in the production, processing, creating high value-added products and marketing of saffron 4. Specialized marketing organizations 5. Target markets and consumer preferences 6. Special brands of saffron for global markets 7. Improved producing and processing of saffron 8. Information center and saffron exchange market 9. Value chain of saffron products 10. Identify products better in order to prevent fraud from bulk exports 11. Accredited exhibitions to introduce saffron products in international markets 12. Saffron products and promotion through reputable media sources throughout the world 13. Internationally recognized standards for producing, processing, and exporting Iranian saffron 14. Legal barriers to prevent bulk export of saffron and rewards for the export of higher value-added products According to Torkamani (2000), saffron growers are not able to deliver their products continuously throughout the year, but the demand for their product is continuous. Some organization must be developed to process and store saffron to meet these needs throughout the year. There is no alliance among saffron producers that organize, buy, sell, distribute, package, and offer marketing services to them. Farmers lack knowledge of market conditions and are subject to large price fluctuations due to supply and demand changes. The formation of local, regional, and national unions for the production, marketing, and exporting of saffron will not only allow additional buyers for saffron but can also help reduce fraud. Saffron producing unions would be responsible for purchasing and collecting saffron from different production centers and delivering services to their farmer members including the provision of fertilizers and credits and promotion of improved production, harvesting, and processing methods. These unions are involved in collecting saffron products and shipping them to packaging, marketing, and exporting centers. Saffron is graded in these centers in a fully sanitary and standardized manner with advanced technologies as value-added products and are packaged and delivered to domestic and foreign markets. In addition to controlling all product standards in the production and packaging processes, the union also carries out other product marketing activities inside and outside the country. This would include appropriate advertising, creating reputable brands, and identifying the desires and needs of consumers. The unions could also segment the market to respond to different demands throughout the world. Hashempour (2009) argued that the most important export problems for saffron are the lack of adequate and appropriate packaging and standardization, the lack of pricing strategies to prevent excessive fluctuations of prices, the lack of extensive and up-to-date advertising on global markets, and the lack of attention to fraud issues. Furthermore, lack of a selection of popular and well-known brands in the global markets, the low use of e-commerce and online sales methods, and inexperienced export service providers are other problems for Iranian saffron. There is the potential to create a powerful brand for saffron. Creating a distinct identity and a credible brand for saffron can increase the bargaining power of traders, increase the price of their products by providing a high-quality product to the world market, and increase the income of producers and traders. Meanwhile, improved, direct connections of producers and traders to the global consumption markets and the elimination of intermediaries can lead to higher producer incomes because markets will be better understood. A review of the literature and related studies indicate that marketing problems are the most important obstacles for increasing the value of saffron export. Therefore, it is necessary to have better insights about marketing methods and challenges for agricultural products such as saffron.

360

SECTION | V Economy and trade of saffron

Jalali et al. (2011) showed that promoting fair competition and reasonable pricing, facilitating access to agricultural markets, providing appropriate infrastructure for agricultural marketing, and establishing agricultural marketing cooperatives play an important role in improving agricultural export markets. In many developing countries, poor marketing strategies make it difficult for agricultural products to thrive (Carter, 1997). This shows the necessity for attention to appropriate marketing strategies in the export of agricultural products. A well-designed marketing strategy has the fundamental goal of increasing sales and achieving a sustainable competitive advantage. Sustainable competitive advantage, as defined by Porter (2008), is achieved through cost management, differentiation of products and services, and a focus on a particular market segment that targets a special group of consumers. While the aim of these strategies is to maximize profit, each of the various strategies applies a different method of maximization. Marketing strategies for achieving competitive advantage involve market penetration, market development, product development, and diversification (Porter, 2008). The application of appropriate marketing strategies to promote agricultural exports is crucial for increasing foreign trade for developing countries. Research suggests that export organizations and companies have found that they can be successful if they use appropriate marketing strategies to achieve sustainable competitive advantage in international markets (Wu et al., 2010). Kashefi et al. (2019) constructed marketing strategies for market penetration, market development, product development, and product differentiation using appropriate indices and investigated their effects on an export performance index of saffron. Their results showed that company experience, the number of employees (or firm size), marketing cost, R&D costs, market development strategy, product development strategy, and differentiation strategy have a significant effect on the export performance of saffron companies. They argue that product development strategy has a higher effect on the export performance index than other variables, and it is recommended that saffron-exporting companies develop and improve their export performance by developing products that are consistent with market demand and conditions. Moreover, developing new product features through R&D and improved technology for production and export of saffron are others recommendations. Due to the positive and significant effect of market development strategy on export performance of saffron companies, developing new markets and partners for exporting saffron is another strategy that could increase saffron exports. Expanding the potential market through new users or new uses of saffron by appropriate marketing and R&D efforts could be very successful. Further, creating value and brand loyalty for saffron could be used as parts of a product differentiation strategy. Moving toward producing healthy and organic saffron products that are matched to international standards can also play an important role. Saffron is produced in a few countries, although more countries have started cultivating saffron. Iran is still the biggest exporter in the world, exporting around 100 tons of saffron each year. The major importers of Iran’s saffron are UAE and Spain. Spain imports Iranian saffron due to its high quality, although they are the second biggest producer of saffron. Table 22.1 shows the exports of different countries during 2011
16. Iran accounted for 78% of saffron exports in 2011, but its share was only 42% in 2016. New competitors such as Afghanistan and Portugal, and a large increase in Spanish exports in 2011, have exposed Iran’s lack of powerful marketing and effective export strategies. Abbasi (1999) showed that Iran’s saffron exports have decreased due to poor marketing efforts and product packaging. In this period, the Spanish share of saffron exports went from 13.4% in 2011 to 29.5% in 2016. Saffron exports by Afghanistan have been increased more than sevenfold between 2011 and 2016, bringing it to third place as its exports are about 20% of Iran’s exports in 2016. Afghanistan is a new and serious competitor for Iran. Aghdaie et al. (2012) showed that firm strategy, structure, and rivalry are important barriers to Iran’s saffron export into international markets. Furthermore, they proposed using appropriate marketing strategies in order to enter international markets with the help of research organizations. Table 22.2 shows saffron imports by the main countries during 2011
16. As shown in Table 22.2, Spain is the leading importer of saffron, and in 2011, its share was 14.5% of total world saffron imports, while in 2016 its share increased to 24.6%. As noted, Spain is also the second largest exporter of saffron in the world accounting for 29.5% of total world saffron exports. Becoming the leading importer and the second leading exporter of saffron, despite producing very little saffron, shows that Spain creates more value-added products in the saffron industry in comparison to other countries. UAE, Italy, and United States are in the next ranking of saffron importers in the world. In the next section, the most important concepts for saffron marketing saffron are reviewed.

22.3

Marketing concepts in the saffron industry

Marketing is about identifying and meeting human and social needs. One of the shortest and best definitions of marketing is “meeting needs profitably.” Managers sometimes think of marketing as the art of selling products, but selling is

Saffron marketing: challenges and opportunities Chapter | 22

361

TABLE 22.1 Exported value of saffron by different countries during 2011
16 in USD. Exporters

2011

2012

2013

2014

2015

2016

World

374,688

194,304

179,045

196,425

215,056

222,781

Iran

292,432

106,000

90,290

99,083

110,432

93,043

50,283

51,423

47,315

47,516

47,160

65,811

Afghanistan

2225

3824

940

3645

3305

16,948

Portugal

4435

3849

10,770

18,056

21,782

11,243

France

6257

2368

3973

7100

6205

6584

Spain

Hong Kong, China

107

209

281

92

894

5208

1034

6589

4222

1067

3231

4097

Greece

928

928

696

1440

1766

3187

China

1204

840

1061

1415

2857

3151

Germany

3391

2645

3353

3038

2177

2033

Switzerland

3316

2159

1498

663

209

2005

India

1516

2171

1885

1850

1605

1386

Other countries

7560

11,299

12,761

11,460

13,433

8085

Netherlands

Source: Data from Trademap, 2015. Trade statistics for international business development. Available from: ,https://www.trademap.org/Index.aspx..

TABLE 22.2 Saffron Imports by leading countries during 2011
16 in USD. Importers

2011

2012

2013

2014

2015

2016

World

266,817

192,336

180,831

196,461

211,938

214,834

Spain

38,715

42,644

38,809

40,139

51,583

53,033

Italy

21,564

19,050

18,365

15,889

17,200

17,446

United States

14,007

14,715

12,553

13,733

13,434

14,938

8353

6133

6944

10,820

9673

14,912

India France

10,650

8669

9427

13,801

10,239

13,319

Sweden

7921

7188

5988

6754

9228

11,097

Argentina

5101

5519

7672

5224

7198

10,922

Saudi Arabia

6733

7946

8286

10,851

11,370

9555

Hong Kong, China

2127

2749

3162

2106

2317

8419

United Kingdom

5353

3764

5844

7325

8174

7003

Switzerland

6910

5603

4812

4183

5287

5327




30,255

21,094

24,286

27,601

5180

Germany

5199

5358

5203

5165

4679

4502

Portugal

2067

1008

1985

4895

4195

3960

Japan

3535

2668

3192

2967

2773

3898

Kuwait

1736

2102

2338

3171

2835

3888

United Arab Emirates

Source: Data from Trademap, 2015. Trade statistics for international business development. Available from: ,https://www.trademap.org/Index.aspx..

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SECTION | V Economy and trade of saffron

FIGURE 22.1 Marketing mix and their elements. Based on Kotler, P., Keller, K.L., 2011. Marketing Management, thirteenth ed., Prentice Hall Inc., Upper Saddle River, NJ.

only the tip of the marketing iceberg. Marketing involves ten main types of things/activities that include goods, services, events, experiences, persons, places, properties, organizations, information, and ideas (Kotler and Keller, 2009). In theory, the marketing planning process consists of analyzing marketing opportunities, selecting target markets, designing marketing strategies, developing marketing programs, and managing the marketing efforts. However, in highly competitive markets, marketing planning is more fluid and is continually refreshed. Companies should continually develop new marketing programs and innovating products, considering customer needs and seeking new advantages and opportunities rather than relying on past strengths. This is especially true when incorporating the Internet into marketing plans. McCarthy classifies various marketing activities into marketing-mix tools, which he calls the four Ps of marketing: product, price, place, and promotion (Kotler and Armstrong, 2010). The marketing variables under each of these are shown in Fig. 22.1. According to Ghodoosi et al. (2016), although Iran is the largest saffron producer in the world, the lack of marketing efforts has left it in a disadvantaged position in this highly competitive market. Iran’s saffron production figures show that in 2012, about 200 tons of saffron was produced in Iran, and that figure increased to 250 tons in 2013. Despite increasing saffron production in Iran, exports of saffron decreased. One of the main reasons for decreasing exports, despite production increases, is the lack of attention to the principles of marketing in the production and export of saffron. This lack of attention has greatly reduced Iran’s share in the global markets. Hence, it is necessary to follow proper marketing principles in this industry if Iran wants to increase saffron export and increase its role and share in global markets. Ghodoosi et al. (2016) examined the role of various marketing dimensions on Iranian saffron exports. They identified the factors affecting saffron exports with an emphasis on marketing mixes. Using the McCarthy model (1964) and the four Ps of marketing, researchers defined each of the marketing mixes for saffron and ranked these elements. They concluded that the saffron product element includes quality, features, design, brand, and packaging. Promotions include advertising, trade fairs, export awards, increased communications, and direct sales that stimulate customer sensitivity and attraction buyers to the product. Place is a set of actions that target the product to the market, including the decisions of marketing managers for the distribution channels of the products. Finally, price is an important feature for saffron buyers. Mohammadi et al. (2017) emphasized that in today’s competitive world, employing marketing concepts and methods while prioritizing the marketing mix approach for products can play an important role in increasing sales and ensuring greater success in the marketplace. Based on the study of Mohammadi et al. (2017) and Ghodoosi et al. (2016), the indices and components of each marketing mixes for saffron are as given in Table 22.3.

22.4

Marketing management tasks for saffron marketing

According to the special features of saffron, Ghorbani (2008) recommended the creation of a regional marketing board for coordinating production, marketing, and exporting activities. The results of saffron marketing board are: G G

Increasing farmer income and production Decreasing production risk

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TABLE 22.3 Marketing mix criteria for saffron exports, including subcriteria. Product

Price

Place (distribution)

Promotion

Saffron products

Price compared to competitors

Quality of transferring the product

Having sale agency in aboard

Organic product

Final price

Extensive distribution network

Special exhibition

Production capacity

Discounts

Distance to importers

Direct selling

Product quality

Inflation

Timely delivery

Sales promotion

Product type

Price list

Importers income

Advertising

Inventory levels

Trade regulation

Exporter consumption Brand

E-commerce

Number of competitors

Packaging

Type of distribution channels

Classifying customers

Fraud in production

Intermediaries management

Guarantee

Market segmentation

Diversity Source: Data from Ghodoosi, M., Mohtashami, T., Motavalli-Habibi, M., Sheddati, S., 2016. Identification and prioritization of marketing mix elements affecting the export of saffron from the perspective of experts. Saffron Agron. Technol. 3, 285
296 (in Persian); Mohammadi, H., Saghaian, S., Alizadeh, P., 2017. Prioritization of expanded marketing mix in different stages of the product life cycle: the case of food industry. J. Agr. Sci. Tech. 19, 993
1003.

G G G G G G G G G G

Decreasing marketing (price) risk Creating insurance products for saffron Creating income stabilization Creating circumstances for a more successful processing industry and better marketing services Controlling more of the saffron market and processing of products Decreasing marketing costs Increasing bargaining power Better attention to preferences and needs of consumers in markets Creating a suitable and up-to-date information system for production and marketing Increasing investment in the agricultural sector especially for saffron The main tasks of a regional marketing board or saffron marketing group (SMG) are:

1. Developing marketing strategies and plans: The first task of a SMG is to identify potential long-term opportunities, given its market experience and core competencies. It must develop concrete marketing plans that specify the marketing strategy and tactics going forward. 2. Capturing marketing insights: A reliable marketing information system to monitor the marketing environment so it can continually assess market potential and forecast demand. Its microenvironment consists of all the players who affect its ability to produce and sell to customers, and competitors. Its macroenvironment includes demographic, economic, physical, technological, political-legal, and social-cultural forces that affect sales and profits. 3. Marketing research system: There needs to be a dependable marketing research system and the SMG could coordinate this. 4. Connecting with customers: The SMG must consider how to best create value for its chosen target markets and develop strong, profitable, long-term relationships with customers. To do so, it needs to understand consumer markets. Who buys saffron and why? What features and prices are they looking for? Where do they shop? The SMG also sells saffron to business markets, including large corporations, professional firms, retailers, and government agencies, where purchasing agents or buying committees make the decisions. The SMG needs to gain a full understanding of the preferences and behaviors of organizational buyers. It needs a salesforce well trained in presenting product benefits. The SMG will not want to market to all possible customers, but instead it must divide the market into major market segments, evaluate each one, and target those it can best serve. 5. Building strong brands: The SMG must understand the strengths and weaknesses of Iranian saffron brands as foreign customers see them. Are its products suitable for each market? Suppose the SMG decides to focus on the consumer

364

6.

7.

8.

9.

SECTION | V Economy and trade of saffron

market and develop a positioning strategy. Should it position itself as a prestigious brand, offering superior products at a premium price with excellent service and strong advertising? Should it build a simple, low-priced product aimed at more price-conscious consumers? Something in between? The SMG must also pay close attention to competitors, anticipating their moves and knowing how to react quickly and decisively to stay competitive. It may want to initiate some surprise moves, in which case it needs to anticipate how its competitors will respond. Shaping the market offerings: At the heart of the marketing program is the product, which includes the product quality, design, features, and packaging. To gain a competitive advantage, the SMG may want to provide leasing, delivery, advising, and training as part of its product offering. A critical marketing decision relates to price. The SMG must decide on wholesale and retail prices, discounts, allowances, and credit terms. Its price should match well with perceived value; otherwise, buyers will turn to competitors’ products. Delivering value: The SMG must also determine how to properly deliver the value embodied in its products to the target market. Channel activities include those the company undertakes to make the product accessible and available to target customers. The SMG must identify, recruit, and link various marketing facilitators to supply its products and services efficiently to the target market. It must understand the various types of retailers, wholesalers, and physical-distribution firms and how they make their decisions. Communicating value: The SMG must also adequately communicate the value embodied by its products and services to the target market. It will need an integrated marketing communication program that maximizes the individual and collective contribution of all communication activities. The SMG needs to set up mass communication programs consisting of advertising, sales, promotions, events, and public relations. It also needs to plan more personal communications, in the form of direct and interactive marketing, as well as hire, train, and motivate salespeople. Creating successful long-term growth: Based on its product positioning, the SMG must initiate new-product development, testing, and launching as part of its long-term view. The strategy should take into account changing global opportunities and challenges. In addition, the SMG should answer some questions for better marketing of saffron such as the following:

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

How can we choose the right market segment(s)? How can we differentiate saffron offerings? How should we respond to customers who buy exclusively based on price? How can we compete against lower-cost, lower-price competitors? How far can we go in customizing offerings for each customer? How can we grow business? How can we build stronger brands? How can we reduce the cost of customer acquisition? How can we keep customers loyal longer? How can we tell which customers are more important? How can we measure the payback from advertising, sales promotion, and public relations? How can we improve salesforce productivity? How can we establish multiple channels and yet manage channel conflict? How can we get the other company departments to be more customer-oriented?

22.5

Conclusion

In many developing countries, agricultural exports cannot find their appropriate markets due to lack of appropriate marketing strategies. Marketing strategies have the fundamental goal of increasing sales and achieving a sustainable competitive advantage for a product. Iran accounts for more than 90% of the world production of saffron but Iranian exporters mainly tend to export saffron in bulk, which minimizes its value-add. The lack of attention to international standards, antiquated distribution systems, and the lack of a central organization that can monitor and cover all the steps of production, advertising, and exporting of saffron creates several challenges. Despite increasing saffron production in Iran, exports of saffron have decreased. One of the main reasons for decreasing exports, despite production increases, is the lack of attention to the principles of marketing in the production and export of saffron that in turn has greatly reduced Iran’s share in the global markets. Hence, it is necessary to follow proper marketing principles in this industry if Iran wants to increase saffron exports and increase its role and share in global markets.

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Creating a regional marketing board for coordinating production, marketing, and exporting activities of saffron is a recommendation for policymakers to export more value-added saffron products into the global markets.

References Abbasi, F., 1999. Difficulties of Iran’s saffron export. Iran Chamber Comm. Monthly J. 384, 37
42 (in Persian). Aghdaie, S.F.A., Roshan, J., 2015. Investigating effective factors on Iran’s saffron exportation. Int. Rev. Manag. Bus. Res. 4 (2), 590. Aghdaie, S.F.A., Seidi, M., Riasi, A., 2012. Identifying the barriers to Iran’s Saffron export by using Porter’s diamond model. Int. J. Mark. Stud. 4 (5), 129. Carter, S., 1997. Global Agricultural Marketing Management. Food and Agriculture Organization of the United Nations, Rome. Ghodoosi, M., Mohtashami, T., Motavalli-Habibi, M., Sheddati, S., 2016. Identification and prioritization of marketing mix elements affecting the export of saffron from the perspective of experts. Saffron Agron. Technol. 3, 285
296 (in Persian). Ghorbani, M., 2008. The efficiency of saffron’s marketing channel in Iran. World Appl. Sci. J. 4, 523
527. Hashempour, M., 2009. Problems of iranian saffron export (M.Sc thesis). Allameh Tabataba’i University, Iran (in Persian). Hosseini, S., Aho-Ghalandari, M., 2007. Analyzing the marketing margins of Iranian saffron. In: The 6th Conference of Iranian Agricultural Economics, 29
30 October 2007, Mashhad, Iran (in Persian). Jalali, S., Mahmoodi, I., Pakravan, M., 2011. Review the state of competitiveness in exporting of Iranian raisins in the global markets. Ag. Dev. J. 86, 49
74. Kashefi, M., Mohammadi, H., Abolhasani, L., 2019. Effect of marketing strategies on export performance of agricultural products: the case of saffron in Iran. J. Agr. Sci. Tech. 21 (4), 910
924. Kotler, P., Armstrong, G., 2010. Principles of Marketing. Pearson Publication, London. Kotler, P., Keller, K.L., 2009. Marketing Management, thirteenth ed. Prentice Hall Inc., Upper Saddle River, NJ. Mohammadi, H., Saghaian, S., Alizadeh, P., 2017. Prioritization of expanded marketing mix in different stages of the product life cycle: the case of food industry. J. Agr. Sci. Tech. 19, 993
1003. Mosavi, S.N., Jokar, M., Faraj-zadeh, Z., 2013. Iran’s market power in the global saffron market. Agric. Econ. Dev. J. 21, 129
149 (in Persian). Pezeshkirad, G., Feali, S., 2010. Challenges and solutions for the processing, marketing and export of saffron: application of Delphi method. Agric. Econ. 4, 137
157 (in Persian). Porter, M.E., 2008. Competitive Strategy: Techniques for Analyzing Industries and Competitors. Simon and Schuster Inc, New York. Torkamani, J., 2000. Economic analysis of production, technical efficiency and marketing of Iranian saffron. J. Sci. Tech. Agric. Nat. Res. 4, 29
45 (in Persian). Trademap, 2015. Trade statistics for international business development. Available from: ,https://www.trademap.org/Index.aspx.. Wu, W.W., Lee, Y.T., Tseng, M.L., Chiang, Y.H., 2010. Data mining for exploring hidden patterns between KM and its performance. Know. Bas. Sys. 23 (5), 397
401.

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Chapter 23

Environmental economic analysis of saffron production Leili Abolhassani1, Soroor Khorramdel2, Michael Reed3 and Sayed Saghaian3 1

Department of Agricultural Economics, Faculty of Agriculture, Ferdowsi university of Mashhad, Mashhad, Iran, 2Department of Agrotechnology,

Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran, 3Department of Agricultural Economics, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, United States

Chapter Outline 23.1 Introduction 23.2 Estimation of potential environmental impacts by lifecycle assessment 23.2.1 Global warming potential 23.2.2 Acidification potential 23.2.3 Aquatic eutrophication potential 23.2.4 Terrestrial eutrophication potential 23.2.5 Aggregated environmental indicator (EcoX) 23.3 Ecological economic analysis of energy use 23.3.1 Ecological energy indicators 23.3.2 Ecological analysis of energy input

23.1

367 367 368 369 369 370 371 372 373 375

23.3.3 Energy economic indicators 23.4 Carbon footprint 23.5 Ecosystem functions and services 23.5.1 Valuation of the ecosystem function 23.5.2 Ecosystem services and impacts from agriculture 23.5.3 Ecosystem services 23.5.4 Environmental impacts 23.6 Green policy analysis matrix of saffron 23.7 Conclusion References

378 380 381 382 382 382 384 385 387 387

Introduction

Since the 1970s, economic studies concerned with environmental issues have been rapidly growing. In environmental economic studies, the economy is considered as a subsystem of the ecosystem, and preserving the environment and natural resources is given as the main focus. Using various theories and empirical techniques, an environmental economic analysis attempts to identify impacts of economic activities on the environment and natural resources and to include them into the financial analysis. In order to achieve this purpose, monetary values of environmental benefits and impacts generated by an economic activity need to be assessed. Since any production causes environmental impacts because inputs are consumed, there are plenty of techniques to determine the most important consequences to the environment. In this chapter, we attempt to describe the most important benefits and impacts of saffron production on the environment. In the first section, we describe studies on the environmental impacts of saffron production. The second section explains energy use and energy indicators of saffron production. In the last part, using the results obtained from the environmental analysis, we reevaluate the economics of saffron production. Since wheat is a plant adapted to almost all types of climatic conditions, assessment of the saffron production system is compared with wheat production.

23.2

Estimation of potential environmental impacts by lifecycle assessment

Lifecycle assessment (LCA) is an appropriate ecological tool for assessing the potential impact of agriculture through evaluations of material and energy flow throughout a product’s lifecycle. LCA presents indicators for policymakers to have measures for improving environmental performance and making modifications to a production system Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00023-X © 2020 Elsevier Inc. All rights reserved.

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(Cherubini, 2010; ISO, 2006). LCA is also considered as a decision-support approach that takes a cradle-to-grave view of activities and highlights the environmental hotspots of production systems (Bartzas et al., 2015; I Canals et al., 2006). Compared to other environmental impact assessment methods, LCA incorporates all the various stages of an agricultural production supply chain (Van Der Werf et al., 2007). LCA has become a widely recognized method for contributing to the development of sustainable farming practices by quantifying the environmental performances of products (Narayanaswamy et al., 2002; Science Applications International Corporation and Curran, 2006). The application of LCA methodology to agricultural systems has improved its ability to provide useful information for decreasing environmental impacts and increasing sustainability of existing agricultural systems (I Canals et al., 2006). The LCA provides numerical scores allowing a comparison of product alternatives relative to climate change, ozone depletion, acidification, eutrophication, toxicity, fossil energy resource depletion, and other environmental impact categories. The LCA is particularly useful in regions of intensive agriculture where the contribution of farming systems to the degradation of the environment is increasingly investigated (BassetMens et al., 2006). All environmental releases, fossil energy resource extractions, and land-use activities that belong to a product lifecycle are translated and aggregated to present an environmental profile in terms of the overall contribution of the product to a number of impact categories (Guinee et al., 2001). Understanding the environmental impacts of agricultural soils in these regions is necessary if we are to improve our knowledge of terrestrial global emissions. Chemical fertilizer is one of the most important inputs in agroecosystems and, thus, the LCA of consuming this input is critical. LCA of fertilizers indicates that despite the technological improvements in its manufacture and use during the last 100 years, greater production intensity increases emissions of pollutants (such as N2O, NOx, NH3, and PO4) contributing to the greenhouse effect, acidification, and eutrophication. Fertilizers containing heavy metals (Cd, Zn, Co, Se, and Hg) also have a toxic effect on water, land, and human beings. The greatest impact on the environment in crop production relying on mineral nitrogen fertilizers is usually associated with changes in land use and eutrophication of aquatic ecosystems (Brentrup and Lammel, 2011; Charles et al., 2006) and in warmer climate conditions with increased global warming potential (GWP) (Fallahpour et al., 2012). The negative environmental impact of the production and use of phosphorus fertilizers is mainly due to the greenhouse effect (transport of raw materials and products) and eutrophication (dispersion of phosphates during fertilizer production and accumulation of phosphogypsum) (Da Silva and Kulay, 2005; Silva and Kulay, 2003). According to the International Organization for Standardization (ISO, 1997) there are four phases in the LCA methodology: (1) objectives and definition of scope, (2) lifecycle inventory (LCI) analysis, (3) lifecycle impact assessment, and (4) integration and interpretation (Brentrup et al., 2004a,b; ISO, 2006). The major applications of LCA are in: (1) investigating the origins of problems related to a particular product, (2) comparing improvement variants of a given product, (3) contributing to sustainable management of agroecosystems, and (4) choosing between comparable products in terms of their impacts on the environment (Guinee et al., 2001; Khoshnevisan et al., 2013a). In the main part of the LCI, the resource consumption (inputs) and emissions (outputs) connected to the system are compiled (Brentrup et al., 2004a). Based on ISO instruction, the impacts, including pollutants to atmosphere, soil, and water, are computed (Finkbeiner et al., 2006). Due to increasing utilization of synthetic chemical inputs in Iranian agriculture and their consequent environmental impacts, LCA seems to be an appropriate method to quantify impacts of various agricultural inputs. Different studies have applied LCA to saffron production. Most of the studies have assessed the environmental impacts of saffron production during its 6- to 7-year product cycle. Comparing the impacts of saffron production with other agricultural products suggests that saffron generates higher environmental impacts. Nevertheless, the environmental impacts of saffron production are dramatically reduced if the impacts are annually computed. In the following analysis we compare the annual environmental impacts of saffron production at different levels of cow manure (0 to more than 150 tons ha21) with environmental impacts produced by wheat (at the levels of 110 to more than 220 kg N ha21) as the other major substitute product (e.g., cultivating wheat in the same area where saffron is produced) (Khorramdel et al., 2017, 2018c; Koocheki et al., 2015; Mollafilabi et al., 2015).

23.2.1 Global warming potential The current rapid rise in global temperature is due to the “enhanced greenhouse effect” from human induced release of GHGs into the atmosphere. The GWP impact defines how much heat can be trapped by greenhouse gas emissions generated when producing a certain product. Carbon dioxide (CO2), nitrous oxide (N2O), and methane (CH4) are the main emissions associated with GWP. N2O emissions from agricultural soils are considered to account for 70%
81% of the increase in the earth’s atmospheric temperature generated by N-based emissions (Bouwman, 1990; Fallahpour et al., 2012). Lal (2004) reported that carbon released to the atmosphere from tillage management is about 55.7 kg ha21 CO2 equivalents.

Environmental economic analysis of saffron production Chapter | 23

369

TABLE 23.1 The global warming potential for irrigated wheat and saffron production systems in Iran. Production system

CO2

N2O

GWP

Irrigated wheata (kg CO2 eq. per 1 ton of seed)

416

16

397

829

88

1397

961

2446

b

Saffron over 7 years (kg CO2 eq. per 1 ton of flower)

CH4

a Data from Khorramdel, S., Rezvani-Moghaddam, P., Aminghafouri, A., 2014. Evaluation of environmental impacts for wheat agroecosystems of Iran by using life cycle assessment methodology. Cereal Res. 4 (1), 27
44 (in Persian). b Data from Khorramdel, S., Abolhassani, L., Rahmati, E.A., 2017. Evaluation of environmental impacts for saffron agroecosystems of Khorasan based on nitrogen fertilizer by using Life Cycle Assessment (LCA). J. Saffron Res. 2, 152
166 (in Persian).

On the other hand, N2O dominates total GWP per ton of grain because 1 kg of N2O has a GWP 310 times higher than that of 1 kg of CO2. Not all GHGs have equal capacity to cause warming, but their impacts depend on the radiative force it causes and the length of time that the gas molecules stay in the atmosphere. Considering these two together, the average warming caused by greenhouse gases is known as the GWP (Pandey et al., 2011). The GWP is expressed in CO2 equivalents derived from the rate of CO2, CH4, and N2O emissions (ISO, 2006). Table 23.1 shows the GWP estimates for producing saffron and wheat systems in Iran. The results show that although the total GWP is higher for the saffron production systems over the 7 years, the annual GWP of the saffron production systems is less than the wheat agroecosystem. The difference is between 60% and 75% depending on the amount of nitrogen used in the production system. CO2 (with 50%) and N2O (with 48%) are the main factors for GWP in wheat production. The main factor for saffron production is CH4 with a 57% share. These greenhouse gas emissions are related to climate and soil conditions, but they are significantly affected by agricultural management (Barton et al., 2008). The main reasons for CO2 and N2O emissions in the wheat production system—as a conventional system—are related to the use of fossil fuel, agricultural machinery, and production and application of N fertilizer (Brentrup et al., 2004a). CH4 emission is mainly generated by manure application, which is the main input in low-input systems. The CH4 emission can vary widely depending on manure management, storage practices (Hansen et al., 2006; Sommer and Moller, 2000), and allocation procedures (Reijnders and Huijbregts, 2005). The fact that management of agricultural systems can influence the three greenhouse emissions was also found by other studies. Moudry´ et al. (2013) declared that N2O and CO2 emissions were significantly higher in conventional agriculture than in organic agriculture.

23.2.2 Acidification potential The impact of acidification potential (AP) is the amount of minerals leached into the soil caused by the uptake of three acids, NH3, NOx, and SO2, being emitted into the atmosphere. The main source of acidifiers on unfertilized fields is fuel (SO2 and NOx) (Brentrup and Lammel, 2011). SO2 primarily originates from combustion of sulfur-containing coal and oil, NOx from combustion processes, and NH3 from animal manure (Brentrup et al., 2004a). LCA of fertilizers takes into account the acidification process (Brentrup et al., 2004a; EEA, 2001). NH3 is closely connected to the handling of farmyard manure and chemical fertilizers. On-farm ammonia mainly volatilizes during application of manure and synthesis of fertilizers. AP grows with increased nitrogen application in the form of NH4 and NO3, mainly due to ammonia volatilization (Table 23.2). The high amount of AP in saffron compared to wheat can be a result of the growing period, which is 1 year for wheat and about 7 years for saffron. The main emissions producing the AP are NH3 (with 87%) and SO4 (with 65%) for wheat production, while NH3 with (48%) and SO4 (36%) are the main emissions for saffron. If cow manure is properly managed in saffron production, the AP impact can be significantly reduced.

23.2.3 Aquatic eutrophication potential Eutrophication is the emission of nutrients, mainly via water but also through the air, which find their way into other ecosystems and affect their relative growth patterns, posing a threat to biodiversity. The impact occurs when the population of microorganisms and algae is overabundant in an aquatic system that causes negative effects on other organisms like fish, birds, and even people. Nitrogen and phosphorous are the main nutrients causing the aquatic eutrophication

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TABLE 23.2 The acidification potential for irrigated wheat and saffron production systems in Iran. Production system

NH3

Irrigated wheata (kg SO2 eq. per 1 ton of seed)

NOx

0.655

b

Saffron over 7 years (kg SO2 eq. per 1 ton of flower)

SO4

0.205

1900.34

AP

0.492

593.856

0.752

1425.25

3919.446

a Data from Khorramdel, S., Rezvani-Moghaddam, P., Aminghafouri, A., 2014. Evaluation of environmental impacts for wheat agroecosystems of Iran by using life cycle assessment methodology. Cereal Res. 4 (1), 27
44 (in Persian). b Data from Khorramdel, S., Abolhassani, L., Rahmati, E.A., 2017. Evaluation of environmental impacts for saffron agroecosystems of Khorasan based on nitrogen fertilizer by using Life Cycle Assessment (LCA). J. Saffron Res. 2, 152
166 (in Persian).

TABLE 23.3 The aquatic eutrophication potential for irrigated wheat and saffron production systems in Iran. Production system

Irrigated wheata (kg PO4 eq. per 1 ton of seed) Saffron over 7 yearsb (kg PO4 eq. per 1 ton of flower)

NH3

NH4

NOx

NO3

0.315

0.297

0.117

0.09

142.273

134.144

52.844

40.649

P

0.572

2591.41

NO3-N

PO4

Aquatic eutrophication potential

0.378

0.001

2.148

12.438

0.059

3144.545

a Data from Khorramdel, S., Rezvani-Moghaddam, P., Aminghafouri, A., 2014. Evaluation of environmental impacts for wheat agroecosystems of Iran by using life cycle assessment methodology. Cereal Res. 4 (1), 27
44 (in Persian). b Data from Khorramdel, S., Abolhassani, L., Rahmati, E.A., 2017. Evaluation of environmental impacts for saffron agroecosystems of Khorasan based on nitrogen fertilizer by using Life Cycle Assessment (LCA). J. Saffron Res. 2, 152
166 (in Persian).

potential (AEP). Emission of NOx and NH3, as well as deposition of phosphorous, plays a significant role in the increasing impact of AEP (Table 23.3). The results show that the AEP is higher in saffron with a 7-year growing period than with the 1-year growing period for wheat. Comparing the AEP in saffron and wheat even for annual impacts, the gap is still huge. In both production systems, the highest share of emissions in the AEP is related to P, which is 27% for wheat and 82% for saffron.

23.2.4 Terrestrial eutrophication potential Emissions of nitrogen, phosphorous, and organic materials enrich soil with nutrients causing excessive growth of microorganisms and plant life (Nilsson and Grennfelt, 1988) known as terrestrial eutrophication potential (TEP). Changes in AEP are mainly determined by the amount of nitrate leaching and phosphorus, which mainly comes from phosphorus fertilizer production (Brentrup et al., 2004b). The application of fertilizers is responsible for almost all the impact within the category eutrophication, mainly as a result of the emissions of the eutrophying components, including NO3, NH3, N2O, NOx, and P (Brentrup et al., 2004a). Therefore, diffuse losses of N and P via leaching, particularly in arable farming with excessive use of chemical fertilizers, may cause eutrophication impacts (Brentrup et al., 2004a). Several studies reported that organic matter can decrease the deposition of P through increases in the availability of soil P content (Afif et al., 1993; Delgado et al., 2002). NOx emission is mainly related to machinery use and translocation (Brentrup et al., 2004a). P is considered as the main cause of aquatic eutrophication (Yang et al., 2008) (Table 23.4). The results show that the annual TEP impact is higher in saffron production than the impact in wheat production. In the wheat production system two emissions, NH3 and NOx, have the same share in the TEP, while in the saffron production system, the emission of NH3 contains 87% of the impact. As observed earlier, NH3 also has a high share in the AP from saffron production. As there is a risk of P leaching through the soil profile and possibly reaching groundwater in soils that are saturated with P, the adoption of manure application rates should be based on crop P requirements.

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TABLE 23.4 The terrestrial eutrophication potential for irrigated wheat and saffron production systems in Iran. Production system

NOx

Irrigated wheata(kg NOx eq. per 1 ton of seed)

NH3

0.492

b

Saffron over 7 years (kg NOx eq. per 1 ton of flower)

TEP

0.492

487.795

0.984

1747.93

2235.725

a Data from Khorramdel, S., Rezvani-Moghaddam, P., Aminghafouri, A., 2014. Evaluation of environmental impacts for wheat agroecosystems of Iran by using life cycle assessment methodology. Cereal Res. 4 (1), 27
44 (in Persian). b Data from Khorramdel, S., Abolhassani, L., Rahmati, E.A., 2017. Evaluation of environmental impacts for saffron agroecosystems of Khorasan based on nitrogen fertilizer by using Life Cycle Assessment (LCA). J. Saffron Res. 2, 152
166 (in Persian).

TABLE 23.5 Aggregated environmental indicator (EcoX) for irrigated wheat and saffron production systems in Iran. Production system

GWP

Acidification

TEP

AEP

EcoX

Irrigated wheat (share per 1 ton of seed)

0.09

0.038

0.02

0.344

0.492

b

0.27

0.05

0.25

0.75

1.32

a

Saffron over 7 years (share per 1 ton of flower) a

Data from Khorramdel, S., Rezvani-Moghaddam, P., Aminghafouri, A., 2014. Evaluation of environmental impacts for wheat agroecosystems of Iran by using life cycle assessment methodology. Cereal Res. 4 (1), 27
44 (in Persian). b Data from Khorramdel, S., Abolhassani, L., Rahmati, E.A., 2017. Evaluation of environmental impacts for saffron agroecosystems of Khorasan based on nitrogen fertilizer by using Life Cycle Assessment (LCA). J. Saffron Res. 2, 152
166 (in Persian).

FIGURE 23.1 Aggregated environmental indicator of saffron agroecosystems in two counties in Khorasan. Data from Khorramdel, S., Abolhassani, L., Rahmati, E.A., 2017. Evaluation of environmental impacts for saffron agroecosystems of Khorasan based on nitrogen fertilizer by using Life Cycle Assessment (LCA). J. Saffron Res. 2, 152
166 (in Persian).

23.2.5 Aggregated environmental indicator (EcoX) In order to compare all environmental impacts related to each production system, normalized values of each impact category are weighted and aggregated to the EcoX. The aim of the final phase of LCA is to analyze the results and formulate conclusions and recommendations in accordance with the goal defined in the first stage (Fallahpour et al., 2012; Kowalski et al., 2007). A higher value of an EcoX score shows a higher environmental impact generated by the related farming activity. Table 23.5 presents the EcoX associated with wheat and saffron production systems. Table 23.5 shows that the total Ecox of saffron production over the 7 years is higher than the EcoX of wheat production, but the annual amount for saffron is about 62% less than wheat. The highest share of EcoX in saffron and wheat production was from AEP with 64% for wheat and 57% for saffron. In most studies conducted on saffron production, aquatic eutrophication (Mollafilabi et al., 2017) has the highest environmental impact. Fig. 23.1 shows the share of each impact category of the EcoX in the two counties of Ghaen and Torbat-e Heydariyeh (two important areas of saffron production) over the 7 years.

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SECTION | V Economy and trade of saffron

FIGURE 23.2 Aggregated environmental indicator of saffron agroecosystems at three nitrogen fertilizer rates. From Mollafilabi, A., Khorramdel, S., Aminghafouri, A., Hosseni, M., 2017. Environmental impacts assessment of saffron agroecosystems using life cycle assessment methodology: (Case study: Torbat-e Heydarieh and Ghaen counties). J. Saffron Res. 4, 229
248 (in Persian).

According to Mollafilabi et al. (2015), although increased use of nitrogen fertilizers raises the EcoX, the share of aquatic eutrophication is the highest in all three levels of nitrogen consumption (Fig. 23.2). The AEP mainly results from using cow manure in saffron production. Therefore, managing cow manure in terms of time of use, amount of consumption, and type of manure can considerably reduce its environmental impact (Feizi et al., 2015).

23.3

Ecological economic analysis of energy use

Resource and energy use is one of the main factors contributing to sustainable agriculture (De Jonge, 2004). In agriculture, energy is an input that is used for various reasons, such as for increasing productivity, enhancing food security, and contributing to rural economic development (FAO, 2000). Traditionally, the efficient use of energy in agriculture was not a high priority; however, the use of energy resources has increased markedly with advanced technology and general agricultural development (Chaudhary et al., 2009). In agriculture, energy consumption has increased in response to increasing population, limited supply of arable land, and a desire for higher living standards. There is now a tendency toward intensive use of energy in agricultural production systems by developing mechanization, and by using chemical fertilizers, high-yielding seeds, and synthetic pesticides. Yet the dependence of conventional agricultural systems on intensive energy is one of the main reasons behind environmental problems such as global warming (Feizi et al., 2015). So it is critical to balance the positive aspects of energy use in increased farm production with the detrimental environmental impacts (Sefeedpari et al., 2014; Taylor et al., 1993). It is also essential to analyze energy use within agriculture through the identification of various energy forms used for a given production level and to measure its environmental influences (Tzilivakis et al., 2005). This type of analysis is conducted using economics (e.g., the energy consumed to produce one unit of a good) and ecology (e.g., the environmental impact of the consumed energy). From an ecological viewpoint, the types of energy used in the production process are an important determinant of its environmental impact. Various energy inputs in the form of human labor, electricity, seeds, fertilizers, diesel fuel, etc., are consumed in the agricultural sector and there have been reports of a high degree of inefficient usage in the literature (Khoshnevisan et al., 2013b; Nassiri and Singh, 2009; Ozkan et al., 2004). All energy used in the agricultural sector is categorized as direct, indirect, renewable, and nonrenewable. The energy used during farming operations is direct energy and indirect energy. Direct energy is referred to as the energy directly consumed in the crop production process (e.g., human labor, electricity, and diesel). Indirect energy consists of the energy embodied in agricultural inputs such as energy used in chemical fertilizers, pesticides, herbicides, fungicides, seed water supply, and farm machinery. Energy inputs can also be renewable and nonrenewable energy. Renewable energy, such as human labor, seeds, and water, are supplied naturally every year, but nonrenewable energy, such as diesel fuel, electricity, chemical pesticides, chemical fertilizers, and machinery, are not supplied every year; they are not reproduced as they are consumed and therefore their supply is limited.

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TABLE 23.6 Quantity of output produced in saffron and wheat production per hectare. Saffrona

Dryland wheatb

Irrigated wheatb

Quantity per hectare

Total energy equivalent

Quantity per hectare

Total energy equivalent

Quantity per hectare

Total energy equivalent

Stigma yield (kg)

3.86

76.43













Leaf yield (kg)

1230

21,402













Corm yield (kg)

1630

24,401.1













Grain yield (kg)







1044.00

15,117.10

2865.00

41,485.20

Straw yield (kg)







1789.70

16,554.90

2578.50

23,851.12

Total energy output (MJ)




45,879.53




31,671.98




65,336.32

a Data from Sahabi, H., Feizi, H., Karbasi, A., 2016. Is saffron more energy and economic efficient than wheat in crop rotation systems in northeast Iran? Sustain. Prod. Consump. 5, 29
35. b Data from Ghorbani, R., Mondani, F., Amirmoradi, S., Feizi, H., Khorramdel, S., Teimouri, M., et al., 2011. A case study of energy use and economical analysis of irrigated and dryland wheat production systems. Appl. Energy 88, 283
288.

From an ecological perspective, wider use of renewable and direct energy sources can make a valuable contribution to meeting sustainable energy targets (Streimikiene et al., 2007). For ecologists, the most important criterion related to energy use is renewability (e.g., the share of renewable energy and the proportion of energy used directly on the farm). Thus, a production system with low dependency on indirect and nonrenewable energies is preferred. For economists, the amount of energy used to produce a product is the most important criterion. For example, with two systems producing the same product, economists would prefer a system with lower energy consumption without considering the type of energy. Energy output
input ratios (total energy output divided by total energy input) from the production process are used to analyze energy efficiency from an economic perspective (Rafiee et al., 2010). Although the two aspects considered by ecologists and economists are somewhat different, they have some common elements. Lower energy use per unit of production is considered by economists to lower environmental impact and to prevent natural resource depletion (Erdal et al., 2007). Agricultural systems with direct and renewable energies, known as low-input agricultural systems, have greater energy efficiency and a higher energy output
input ratio. This is desirable (Dalgaard et al., 2001). In the following section, we present an evaluation of saffron production compared to wheat production using energy indicators from economical and ecological aspects. Some indicators assess the production process by considering only ecological concerns. We call them “ecological energy indicators.” Others use both energy and economic factors; we call them “energy economic indicators.” Outputs produced through saffron and wheat production are presented in Table 23.6, and inputs used are given in Table 23.7. The saffron and wheat energy ratios—known as economic-energy indicators—are presented in Section 23.3.3. There are other studies that measure inputs consumed for saffron and wheat productions. For instance, the study conducted by Khanali et al. (2016) calculates total energy used for the saffron production system at 99,236 MJ ha21. The energy output
input ratios are 0.43, 3.4, and 1.4 for saffron, dryland wheat, and irrigated wheat, respectively. The main energy inputs are seeds (corms) with 60% for saffron, diesel fuel with 45% for dryland wheat, and nitrogen with 33% for irrigated wheat. One unit of energy used in saffron production yields 0.42 units of energy output with 53% in the corm and about 46% in the leaf.

23.3.1 Ecological energy indicators Ecological indicators are more concerned with the quality of energy movement from primary sources to high-level consumers. The related indicators specify changes in the status of the environment during the energy transfer. This depends highly on which types of energy sources have been used over the production process. The following indicators are used to assess farm production from the ecological aspect. Direct
indirect energy ratio: A high ratio of direct energy compared to indirect energy indicates that the farming system may use more labor and less machinery with simple technology (Liu et al., 2011). Therefore, a production

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SECTION | V Economy and trade of saffron

TABLE 23.7 Quantity of input consumption in saffron and wheat production. Saffrona

Dryland wheatb

Irrigated wheatb

Quantity per hectare

Total energy equivalent

Quantity per hectare

Total energy equivalent

Quantity per hectare

Total energy equivalent

Human labor (h)

1831.71

3571.83

34.5

67.27

108.00

210.60

Machinery (h)

21.46

1345.36

10.50

658.35

18.00

1128.60

Diesel fuel (L)

99.88

5016.86

83.90

4214.30

218.00

10,950.14

Nitrogen (kg)

87.4

6595.204

25.00

1886.50

200.00

15,092.00

Phosphate (P2O5) (kg)

75.9

992.013







70.00

914.90

Potassium (K2O) (kg)

47.5

529.625







75.00

836.25

1

280.44

1.00

280.44

Pesticide (L) Herbicides (kg or L)

2

476







Topic (L)













1.00

271.38

2,4-D (L)













1.5

127.36

Manure (kg)

48,000

14,400













Fungicide (kg or L)

5

460

0.20

36.38

0.50

90.95

Electricity (kWh)

21,513.4

5976







1200.00

4320.00

Water for irrigation (m3)

3850

3927







6000.00

6120.00

Seed (kg)

4300

64,371

110.00

2211.00

250.00

5025.00

107,660.9




9354.20




45,367.62

Total energy input (MJ)

a Data from Sahabi, H., Feizi, H., Karbasi, A., 2016. Is saffron more energy and economic efficient than wheat in crop rotation systems in northeast Iran? Sustain. Prod. Consump. 5, 29
35. b Data from Ghorbani, R., Mondani, F., Amirmoradi, S., Feizi, H., Khorramdel, S., Teimouri, M., et al., 2011. A case study of energy use and economical analysis of irrigated and dryland wheat production systems. Appl. Energy 88, 283
288.

system with a high direct energy ratio may indicate a more sustainable system due to less machinery inputs. The direct and indirect energy used to produce saffron and irrigated wheat is given in Table 23.8. The direct ratio is higher for producing irrigated wheat than saffron, suggesting that irrigated wheat production is more sustainable. The high share of seed in terms of total energy equivalent is the main reason for the small direct ratio for saffron. Renewable
nonrenewable energy ratio: Environmental impacts, such as global warming emissions and pollution, caused by turning resources into energy are higher for nonrenewable energy than renewable energy. Therefore, in a production process, consumption of more renewable energy compared to nonrenewable energy leads to lower environmental impacts and less environmental damages, indicating more sustainable energy transfer (Streimikiene et al., 2007). The ratio of renewable to nonrenewable energy is used to determine the sustainability of the production process. A product with a high renewability ratio would be ecologically sustainable. Table 23.8 shows the ratio calculated for saffron and wheat production. The ratio estimated for saffron production is 12 times more than the ratio for wheat production. This implies that saffron production is more sustainable than wheat production from an ecological viewpoint of energy use because saffron uses resources with more long-term availability and less environmental impact.

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TABLE 23.8 Direct, indirect, renewable, and nonrenewable energies used for wheat and saffron production. Types of energy

Saffrona

Irrigated wheatb

Dryland wheatb

MJ ha21

%

MJ ha21

%

MJ ha21

%

Direct energy

18 491.69

17.14

21600.74

47.61

4281.57

45.77

Indirect energy

89 409.20

82.86

23766.89

52.39

5072.67

45.23

Ratio (direct/indirect)

0.21

0.91

1.01

Renewable energy

86 269.83

79.95

11355.6

25.03

2278.27

24.35

Nonrenewable energy

21 631.07

20.05

34012.03

74.97

7075.97

75.71

Ratio (renewable/nonrenewable)

3.99

0.33

0.32

a Data from Sahabi, H., Feizi, H., Karbasi, A., 2016. Is saffron more energy and economic efficient than wheat in crop rotation systems in northeast Iran? Sustain. Prod. Consump. 5, 29
35. b Data from Ghorbani, R., Mondani, F., Amirmoradi, S., Feizi, H., Khorramdel, S., Teimouri, M., et al., 2011. A case study of energy use and economical analysis of irrigated and dryland wheat production systems. Appl. Energy 88, 283
288.

FIGURE 23.3 Share of total energy used in irrigated wheat. Data from Ghorbani, R., Mondani, F., Amirmoradi, S., Feizi, H., Khorramdel, S., Teimouri, M., et al., 2011. A case study of energy use and economical analysis of irrigated and dryland wheat production systems. Appl. Energy 88, 283
288.

23.3.2 Ecological analysis of energy input Human labor, fertilizers, machineries, and water are the main energy sources for crop production. Here we analyze the total energy and shares of each energy source to produce saffron compared to wheat. Figs. 23.3
23.5 show the resources consumed in saffron and wheat production. Seed, fertilizer, and human labor are the three inputs with the highest shares of total energy used for saffron production, while nitrogen, diesel fuel, and water are the highest shares of inputs for irrigated wheat production. For dryland wheat, diesel fuel, seed, and nitrogen are the main sources of energy input. The study conducted by Khanali et al. (2016) suggested that fertilizers dominate total energy consumption for saffron production. Comparing the inputs used for the production of wheat and saffron, it is obvious that wheat has a mechanized system, while saffron is a laborintensive activity. Furthermore, it is obvious that wheat uses more energy sources that are in critically short supply, such as water and fuel. It is interesting to note that according to Sahabi et al. (2016), energy use related to water is about 64% more for wheat cultivation than for saffron production. Therefore, saffron is suggested for regions facing water scarcity. Moreover, the environmental impacts of nitrogen (the main fertilizer for wheat) are higher than the impacts of manure (the main fertilizer in saffron cultivation). Considering energy the assessment ratios in Table 23.8 saffron could dominant wheat in terms of sustainable if it would reduce its use of indirect energy, such as fuels, and replace them with greener inputs such as biofuels. Saffron’s renewable energy ratio is much higher (3.99 vs 0.33 for dryland wheat), so its production is much greener from this perspective. Fig. 23.6 shows the differences between the energy used in saffron and wheat production. Some types of energy, such as manure, which is used only for saffron production, are not included in the graph.

376

SECTION | V Economy and trade of saffron

FIGURE 23.4 Share of total energy used in dryland wheat. Data from Ghorbani, R., Mondani, F., Amirmoradi, S., Feizi, H., Khorramdel, S., Teimouri, M., et al., 2011. A case study of energy use and economical analysis of irrigated and dryland wheat production systems. Appl. Energy 88, 283
288.

FIGURE 23.5 Share of total energy used in saffron. Data from Sahabi, H., Feizi, H., Karbasi, A., 2016. Is saffron more energy and economic efficient than wheat in crop rotation systems in northeast Iran? Sustain. Prod. Consump. 5, 29
35.

FIGURE 23.6 Difference in energy used between saffron and wheat production per hectare. Data from Sahabi, H., Feizi, H., Karbasi, A., 2016. Is saffron more energy and economic efficient than wheat in crop rotation systems in northeast Iran? Sustain. Prod. Consump. 5, 29
35.

Much higher levels of seed, fungicide, and human labor are consumed in saffron production. Among the common indirect energy sources, seed and fungicides were the two sources used more intensively in saffron production over the 7-year period than in wheat over 1 year. There is also a huge difference between saffron and wheat production in terms of human labor. Saffron is known as a labor-intensive product (Bakhtiari et al., 2015); some studies label it as a “family farming” production system (Koocheki et al., 2017). Saffron cultivation in the studied area is not mechanized and human labor plays a key role in handling all agricultural operations. Mechanized operations (plowing, weeding, and

Environmental economic analysis of saffron production Chapter | 23

377

spraying) are only performed during the preparation process. Almost all of the farming activities in saffron production use human labor exclusively in the production process. For example, 1 hectare of a saffron farm requires 17 times more workers than a hectare of an irrigated wheat farm (Table 23.7). Fig. 23.7 shows the energy used in saffron production between mechanized operations and human labor. The share of each farming activity in the total labor input is shown in Fig. 23.8. A considerable amount of human labor is employed for planting and agronomic management of saffron, including hand weeding, flower harvesting, and stigma separating. According to Khanali et al. (2016), most human labor is involved with harvesting and separating stigmas (an average of 7152.1 MJ ha21). Although traditional, family-oriented saffron farming requires a considerable amount of human labor, particularly for harvesting, the introduction of automated machines for cutting saffron flowers (Gracia et al., 2009) and for separating stigma (Emadi and Yarlagadda, 2008) will likely reduce human labor use in the near future. This means that energy management and field situations clearly affect total energy consumption in the saffron cultivation system. The last stage of energy analysis, consumption of fertilizers and chemical inputs such as pesticide and micronutrients, was also analyzed over the 6 years of the saffron growing period. The average N-based fertilizer consumption in the region for the period of 6 years is estimated at 63,737 MJ ha21 while it ranges from 42,304 to 117,1150 MJ ha21 depending on the farm management system. The average consumption of P- and K-based fertilizers is estimated at 4589.8 and 1459.4 MJ ha21, respectively. This shows that N-based fertilizer (with the share of more than 90%) is the major source of energy consumption. Of the total N-, P-, and K-based fertilizers consumed in saffron cultivation, 11%, 36%, and 27% were consumed during land preparation, respectively, and the rest was consumed during the whole lifecycle of the crop (Khanali et al., 2016) (Fig. 23.9). The application of

FIGURE 23.7 Mechanized and nonmechanized energies in saffron production. From Khanali, M., Movahedi, M., Yousefi, M., Jahangiri, S., Khoshnevisan, B., 2016. Investigating energy balance and carbon footprint in saffron cultivation
a case study in Iran. J. Clean. Prod. 115, 162
171.

FIGURE 23.8 Distribution of human labor energy consumption in saffron production. From Bakhtiari, A.A., Hematian, A., Sharifi, A., 2015. Energy analyses and greenhouse gas emissions assessment for saffron production cycle. Environ. Sci. Pollut. Res. Int. 22, 16184
16201.

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SECTION | V Economy and trade of saffron

FIGURE 23.9 Energy consumption for fertilizers, pesticides and micro nutrient in a 6-year period of saffron production. From Khanali, M., Movahedi, M., Yousefi, M., Jahangiri, S., Khoshnevisan, B., 2016. Investigating energy balance and carbon footprint in saffron cultivation
a case study in Iran. J. Clean. Prod. 115, 162
171.

all types of fertilizers in saffron cultivation is based on farmers’ experience, not what is recommended (Khoshnevisan et al., 2013b; Mohammadi et al., 2014; Mousavi-Avval et al., 2011). The timeframe for chemical pesticide consumption is different, with 13% of the total consumption occuring during farm preparation. Although there was no pesticide usage in the first year, there was an increasing trend toward pesticide application up to the fifth year, which accounts for 19% of consumption (Khanali et al., 2016).

23.3.3 Energy economic indicators Energy economic indicators assess the production process through energy inputs consumed for production in either amount or value of the final product. The following indicators are used to assess saffron production from energy economic aspects with comparisons to wheat. Net energy refers to the difference between energy stored in the inputs and the energy contained in the final outputs. The following formula is used to calculate this indicator: Net energy ðMJ ha21 Þ 5 energy output ðMJ ha21 Þ 2 energy input ðMJ ha21 Þ

(23.1)

Comparing this indicator for saffron and wheat (Table 23.8) shows that this indicator is 8%
21% higher in saffron than wheat. This indicates that saffron is more efficient in converting primary energy to consumable energy. Energy use efficiency describes the ratio of energy input to energy output. This indicator is estimated by the following formula: Energy use efficiency 5

EO EI

(23.2)

where EI and EO are energy input and energy output based on MJ per hectare. A high value for this indicator suggests that either much energy is obtained from a given energy input or little energy input is used to obtain a given output. Specific energy refers to the energy used for producing one unit of production. To calculate this indicator for an agricultural activity, the following formula is used: S5

EI P

(23.3)

where S denotes specific energy, EI is energy input (MJ per hectare), and P denotes amount of production yield (kg per hectare). High values of specific energy show that a considerable amount of energy has been lost during the production

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379

process (Chauhan et al., 2006). Table 23.8 presents values of this indicator for saffron and wheat production, indicating that the amount of energy used to produce one unit of saffron is more than for wheat. Energy intensiveness is another measure of energy efficiency in a production process. This indicator is calculated as units of energy per unit of an economic variable such as production cost or net income. Here we consider three types of energy intensiveness: per unit of production cost, income, and net benefits for saffron and wheat (Table 23.8). The following formulas are used to calculate this indicator: IE CC IE Energy intensiveness of income 5 IC IE Energy intensiveness of net benefit 5 NB Energy intensiveness of cost 5

(23.4) (23.5) (23.6)

where IE, CC, IC, and NB denote energy input, cost of cultivation, income from cultivation, and net benefit of cultivation, respectively, measured in dollars per hectare. High energy intensity of cost indicates that more energy is consumed during the production process. High values of energy intensity of income or net benefit indicate that a high amount of energy is required to produce one unit of income or net benefit. In general, energy intensiveness for saffron production is low indicating that little energy is required in terms of cost, or to produce one dollar of income or net benefit. Energy intensiveness in irrigated and dryland wheat is between 4 and 20 times higher than in saffron production. Energy productivity is used to evaluate energy efficiency of production. This indicator is calculated by: Energy productivity 5

Y IE

(23.7)

where Y denotes yield measured as kg per hectare and IE is input energy calculated based on MJ per hectare. Energy economic productivity is used to evaluate economic efficiency of production from the energy aspect. This indicator is calculated by: Energy economic productivity 5

V IE

(23.8)

where V is value added measured on dollars per hectare and IE is input energy as MJ per hectare. The last two rows of Table 23.9 present productivity estimates for saffron and wheat production. Wheat production has higher energy productivity than saffron, but if the production process is evaluated based on monetary value of

TABLE 23.9 Energy economic indicators for saffron and wheat. Indicators

Saffrona (based on stigma)

Dryland wheatb (based on grain)

Irrigated wheatb (based on grain)

Net energy (MJ ha21)

24,299.35

19,968.69

22,317.73

1.44

3.38

5590.72

15.83

8.96

1

46.58

79.27

3.82

18.22

40.28

Energy intensiveness (Net benefit: MJ $ )

4.03

29.93

81.88

Energy productivity (kg MJ21)

0.00018

0.053

0.112

2.48

0.012

0.033

Energy use efficiency

0.0035 21

Specific energy (MJ kg ) 21

Energy intensiveness (Cost: MJ $ ) 21

Energy intensiveness (Income: MJ $ ) 21

21

Energy economic productivity ($ MJ )

a Data from Sahabi, H., Feizi, H., Karbasi, A., 2016. Is saffron more energy and economic efficient than wheat in crop rotation systems in northeast Iran? Sustain. Prod. Consump. 5, 29
35. b Data from Ghorbani, R., Mondani, F., Amirmoradi, S., Feizi, H., Khorramdel, S., Teimouri, M., et al., 2011. A case study of energy use and economical analysis of irrigated and dryland wheat production systems. Appl. Energy 88, 283
288.

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SECTION | V Economy and trade of saffron

output (energy economic productivity), saffron production is more economically viable and preferred to wheat production. There have been other estimates of energy indices for saffron and they depend on the inputs used and the production process. For example, Khanali et al. (2016) conclude that the energy ratio, energy productivity, and net energy for saffron production are 0.0044, 0.0003 kg MJ21, and 298,818.5 MJ ha21, respectively. Putting all this information together, the conclusion is that saffron production has a little benefit if it is evaluated by ecological indicators such as the amount of output (kg) per hectare. When considering the monetary value of production, saffron is more beneficial because the environmental benefits from saffron production are much higher than environmental costs (see Sections 23.5 and 23.6). This is because saffron is not a bulky product, so its product yield in the form of energy (MJ) and mass (kg) is very small compared to other crops such as wheat. However, the small amount of energy produced by saffron stigma, the tradable part of saffron, generates huge economic value.

23.4

Carbon footprint

Production, storage, and distribution of inputs and usage of machinery involve combustion of fossil fuels and the use of this energy emits CO2 and other GHGs into the atmosphere (Lal, 2004). In total, agriculture worldwide emits between 5.1 and 6.1 Pg CO2 eq. year21, accounting for 10%
12% of worldwide GHG emissions. These emissions are principally in the form of methane (CH4) from animal production (3.3 Pg CO2 eq. year21); nitrous oxide from land cultivation (2.8 Pg CO2 eq. year21); and carbon dioxide from soil carbon changes and energy utilization (0.04 Pg CO2 eq. year21) (Bonesm et al., 2012). We measure all gases in carbon dioxide equivalents (Emadi and Yarlagadda, 2008; Smith et al., 2013). A crop’s carbon footprint is defined as the carbon emission or carbon consequences from agricultural practices in the production chain (Burne et al., 2010; Hillier et al., 2009; West and Marland, 2002). To calculate the carbon footprint for a given activity, LCA is used. We compare the carbon footprint of producing saffron compared to wheat (Table 23.10). Total greenhouse gas emission in saffron production is calculated as 10,897 kg CO2 eq. ha21 over a period of 6 years. So saffron cultivation in this region is responsible for 1816 kg CO2 eq. ha21 year21 (Khanali et al., 2016). This emission estimate is associated with on-farm (to produce final products) and off-farm (to produce inputs) activities. Wheat production in Iran generates an average of 6579.19 kg CO2 eq. ha21 year21, which is equal to 0.27 kg CO2 eq. ha21 year21 for producing 1 kg of wheat (Bakhshaei, 2016). This amount is different from area to area, and depends on climatic conditions, edaphic characteristics, and management. For instance, in the Khozastan province, located in the

TABLE 23.10 On-farm and off-farm carbon footprint for saffron production. Input

Unit

Off-farm emissions

kg

GWP (kg CO2 eq. per ha)

N-based fertilizers

kg

2750.3

P-based fertilizers

kg

835.1

K-based fertilizers

kg

127.8

Pesticides

kg

91.3

Farmyard manure

t

2713.7

Diesel fuel

l

27.6

On-farm emissions Direct N2O from fertilizers

kg

3796

Direct N2O from manure

kg

385.4

Diesel fuel

kg

169.8

Total GHG emissions

kg CO2 eq.

10897

Source: From Khanali, M., Movahedi, M., Yousefi, M., Jahangiri, S., Khoshnevisan, B., 2016. Investigating energy balance and carbon footprint in saffron cultivation
a case study in Iran. J. Clean. Prod. 115, 162
171.

Environmental economic analysis of saffron production Chapter | 23

381

FIGURE 23.10 Share of inputs in greenhouse emissions from wheat production. From Bakhshaei, S., 2016. Investigation of Carbon Footprint for Some Crops of Iran (Ph.D. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

east-south of Iran (Moslem et al., 2015), the amount of greenhouse emissions is 7541.04 kg CO2 eq. ha21 year21, which is almost three times the amount generated by 1 hectare of saffron over 7 years (e.g., 2325.5 kg CO2 eq. ha21) (Bakhtiari et al., 2015). Chemical fertilizers have the most share in generating greenhouse emissions for wheat (Fig. 23.10). Wheat production generates 3.6% more greenhouse gases (per hectare) than saffron production.

23.5

Ecosystem functions and services

Agriculture plays a crucial role in producing food for consumers and earning income for farmers. But it also plays an important role in the provision of ecosystem goods and services. Ecosystem services are “the conditions and processes through which natural ecosystems, and the species that make them up, sustain and fulfill human life” (Daily, 1997). Another definition for ecosystem services are “the set of ecosystem functions that are useful to humans” (Kremen, 2005). De Groot (1992) defines ecosystem functions as “the capacity of natural processes and components to provide goods and services that satisfy human needs, directly or indirectly.” The provision of ecosystem services and subsequent benefit to humans is underpinned by a series of biophysical processes and ecological functions, which themselves are driven by biological diversity (Balvanera et al., 2006). Ecosystem functions result from the interactions among characteristics, structures, and processes (Turner et al., 2000) constituting the physical, chemical, and biological exchanges and processes that contribute to the selfmaintenance and self-renewal of an ecosystem (e.g., nutrient cycling and food-web interactions). Ecosystem functions involve interactions between biotic and abiotic system components in achieving any and all ecosystem outcomes (National Research Council, 2005). Based on the Millennium Ecosystem Assessment (MEA, 2005), ecosystem functions can be conveniently grouped into four categories of: “provisioning function,” “regulating function,” “habitat or supporting function,” and “cultural function.” Regulatory function refers to benefits achieved by regulation of ecosystem processes including gas and nutrient exchange, disturbance prevention, water regulation, soil retention and formation, waste treatment, pollination, and biological control. Human life is impossible without the regulatory function. Habitat function is the provision of habitat and maintenance of biological diversity. Habit services of the environment provide habitat for migratory species and “gene-pool” protectors (Lead et al., 2010). Provisioning function includes the production of food and other raw materials such as medicinal, genetic, and ornamental resources. Obviously human life is impossible without the provisioning function. Cultural function refers to esthetic, recreational, cultural, and spiritual functions. This classification concept was established in order to integrate the concept of ecosystem services and values into landscape planning, management, and decision making (De Groot et al., 2010). Fig. 23.11 shows some details on each category of the four ecosystem functions.

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SECTION | V Economy and trade of saffron

FIGURE 23.11 Main categories of ecosystem functions. Based on MEA, 2005. Ecosystems and Human Well-Being: Synthesis. Island Press, Washington, DC.

23.5.1 Valuation of the ecosystem function Valuation for ecosystem services is rapidly becoming a major consideration in the protection of natural resources within production landscapes (Rapidel, 2011). The classification of ecosystem services and functions is a great contribution to their economic valuation (De Groot et al., 2002; National Research Council, 2005). Such a classification of ecosystem services, in addition to providing a systemic way of analyzing ecosystem services, allows comparisons and trade-offs among the full range of benefits reflecting human well-being from ecosystem services. Assigning monetary value to ecosystem services can aid in making environmental problems visible, and thus contribute to the process of decision making (Spangenberg and Settel, 2010; Wilson and Howarth, 2002). Many methods have been applied to estimate the economic value of ecosystem services. All methods can be classified with three approaches: “revealed willingness to pay,” “imputed willingness to pay,” and “stated willingness to pay.” In the revealed preference method, observed behaviors are used to estimate the monetary value of environmental functions. In the imputed preference method, the value of ecosystem services are measured by computing the cost that either people are bearing if the ecosystem services are discontinued or what they are willing to pay in order to avoid the adverse effects that would occur in the absence of the ecosystem services. In the stated preference method, people’s answers to survey questions are used to estimate the value of ecosystem services. The interested reader is referred to Champ et al. (2003) for further description on willingness-to-pay calculations.

23.5.2 Ecosystem services and impacts from agriculture Agricultural systems have been primarily considered as sources of provisioning services. However, their contributions to other types of ecosystem services are increasingly recognized (MEA, 2005). A range of regulating and cultural services, in addition to the provision services, can be provided by agricultural systems during the ecosystem process. Regulating services gained by agriculture can be flood control, carbon storage, pest control, and genetic diversity for future agricultural use. Cultural services may be included as education, recreation, and tourism. Whether a particular ecosystem service is provided for a given agricultural system or how much of such a service is generated depends on management practices, such as crop diversity and field size (Power, 2010). Although agriculture contributes to improving the environment, it can also have negative impacts. Pollution resulting from using pesticides and chemical fertilizers are the most important externalities generated from agricultural activities. As discussed earlier, GWP, AP, AEP, and TEP are the main negative environmental impacts of agriculture on the environment. The net share of agriculture in the world’s emission of greenhouse gas is about 14% (Cooper et al., 2011). Estimating monetary costs for these environment contributions make the negative consequences of farming activities visible, and thus provide a more wholistic assessment of farming systems. The graph in Fig. 23.12 presents potential ecosystem services and impacts that agricultural systems can have on the environment. In the following section, we review studies that estimate economic values of various environmental functions as well as negative environmental impacts generated during saffron production. We also measure the environmental impacts for wheat production in order to compare the two systems. We reconstruct the policy matrix analysis (PAM) previously presented (in Section 21.9) by including the environmental costs and benefits generated by saffron production. We call this matrix the “green PAM.”

23.5.3 Ecosystem services The four ecosystem services of carbon sequestration, oxygen production, food production, and informational function are provided by saffron farms.

Environmental economic analysis of saffron production Chapter | 23

383

FIGURE 23.12 Potential ecosystem functions and impacts generated by an agricultural system. From Power, A.G., 2010. Ecosystem services and agriculture: tradeoffs and synergies. Philos. Trans. R. Soc. Lond. B Biol. Sci. 365, 2959
2971.

FIGURE 23.13 Carbon sequestration of aboveground and belowground tissues of saffron. From Khorramdel, S., Mollafilabi, A., Latifi, H., 2018a. Evaluating the potential of carbon sequestration and global warming potential for saffron fields (Case study: Khorasan-e Razavi Province). J. Plant Prod. Res. 25 (1), 13
29 (in Persian).

Carbon sequestration is the process through which atmospheric carbon is captured and stored for the long term. The process slows the atmospheric accumulation of greenhouse gases released by such activities as burning fossil fuels. Plants can play an important role in this process. The amount of carbon sequestrated by saffron is 1.57 tons ha21 from aboveground tissues and 6.26 tons ha21 from belowground tissues. The shares of carbon sequestration for the saffron parts are corm (74%), tunic (18%), leaf (6%), and flower (2%) (Khorramdel et al., 2018a). The amount of carbon sequestration varies depending on the climate and time of year. Figs. 23.13 and 23.14 presents carbon sequestration by different tissues of saffron and wheat (Khorramdel et al., 2018a,c). The two plants play a similar role in storing atmospheric carbon. Wheat sequesters 8.25 tons ha21 with 96% of it from aboveground tissues and 4% from belowground tissues (Khorramdel et al., 2018c). Oxygen production is another important ecosystem function performed by plants and is an essential component of farming services. Saffron production generates 1.2 kg of oxygen per kg of agricultural dry matter. According to Thornes (2010) this oxygen has a value of $2.70. The average yield for saffron is 3.7 kg of dry stigma per hectare, so the amount of oxygen produced during saffron growth is 4.44 kg ha21 with a value of $11.99 dollars (based on 2009 prices). Food production: The average saffron yield in Iran is 3.7 kg ha21 and the price of 1 kg of Iranian saffron was $1500 in 2016, so the value of saffron production was $5500 per hectare in 2016. With the same price for 1 kg21 of dry stigma, Khorramdel et al. (2018b) calculated the value of saffron production located in the Khorasan Razavi between

384

SECTION | V Economy and trade of saffron

FIGURE 23.14 Carbon sequestration of aboveground and belowground tissues of wheat. From Khorramdel, S., Shabahang, J., Ahmadzadeh-Ghavidel, R., 2018c. Evaluation of carbon sequestration and global warming potential of wheat in Khorasan-Razavi province. Agritech 38 (3), 234
240.

$1617 and $2399 per hectare with an average of $2008. Their estimate for the value of wheat production was calculated at $499 per hectare (Koocheki et al., 2015). For more details on the production economics of saffron see Chapter 21, Economic analysis of saffron production. Biogenetic resources: Biodiversity is one of the main elements of habitat function. This term refers to the life variation in the soil including variations among plants genes and species. Farming activities can either contribute or harm biodiversity depending very much on the intensity of agricultural practices and pesticide usage (Mclaughlin and Minea, 1995). Due to low usage of water, hand cultivation, and manure fertilization, saffron is considered as an ecofriendly plant (Small, 2016). In the Khorasan Razavi province, the economic value of saffron biodiversity—based on 15% of the total environmental value—is between $637 and $2466 (with an average of $986) (Khorramdel et al., 2018b). It is $228 for wheat farms in the province (Koocheki et al., 2015). Informational function: Many saffron farms are open to the public during harvest season. As tourism has been increasing due to saffron festivals, the importance of saffron’s cultural services has been growing. The value of cultural services was estimated to be between $328 and $1803.6 (with an average of $986.6) per hectare for saffron farms located in Khorasan Razavi province (Khorramdel et al., 2018b). The cultural value was very low for wheat farms (Koocheki et al., 2015).

23.5.4 Environmental impacts Emission of greenhouse gases: Saffron farms in Iran produce 3.7 kg of stigma per 1 hectare. One ton of stigma generates between 1444.5 and 2503 kg of greenhouse gases during 7 years of growth. If the cost of carbon is $14.25 per ton, the cost of saffron’s greenhouse gas emissions is between $37.41 and $64.83. Khorramdel et al. (2018b) estimated that greenhouse gas emission costs were between $272.6 and $618 (averaging $446.33) per hectare in the Khorasan Rzavi. Greenhouse gas emission costs were estimated to be $175.3 per hectare for wheat farms (Koocheki et al., 2015). Nutrient pollution: Animal manure and fertilizers that are rich in nitrogen and phosphorus are the main sources of nutrients. The amount of nutrient pollution is estimated by plant uptake efficiency and degree of fertilizer solubility. Considering that the degree of solubility is higher for nitrogen than phosphorous, the amount of nutrient pollution produced by nitrogen fertilizers is higher than the amount produced by phosphorous fertilizer. Based on the study of Norris et al. (2010) that considered $1.60 as the cost per kilogram for nutrient pollution, the cost was estimated to be from $135.7 to $172.7 with an average of $155.33 for 1 hectare of saffron (Khorramdel et al., 2018b), while it was $40 per hectare for wheat farms (Koocheki et al., 2015). Fig. 23.15 presents the share of each ecosystem service benefits and environmental costs relative to the net ecosystem value. Considering the average values, the share for the food function is the highest. Notice that the positive environmental services generated by saffron production are much higher than the environmental costs. By comparison, the environmental benefits generated by wheat farms in Khorasan Razavi province was $2171 per hectare on average with the highest share from oxygen production. The environmental cost was calculated at $215 per hectare with most costs coming from CO2 production. The net environmental value was $1956.6 per hectare, which is 57% lower than the net value calculated from saffron production for the same region. Aggregating all the information in this section together, the ecosystem services generated by saffron production is valued at $5154 per hectare, while the environmental impact costs are valued at $6016 per hectare. Ecosystem services from saffron production are about eight times more than the environmental costs generated from using fertilizers.

Environmental economic analysis of saffron production Chapter | 23

385

FIGURE 23.15 Ecosystem services and environmental costs from saffron production. Data from Khorramdel, S., Rezvani-Moghaddam, P., Aminghafouri, A., 2018b. Economic evaluation of agroecosystem services of saffron in Khorasan Razavi province. Saffron Agron. Technol. 6 (1), 73
89 (in Persian).

FIGURE 23.16 Ecosystem services and negative impacts of saffron farms of various sizes in Khorasan Razavi. From Khorramdel, S., Rezvani-Moghaddam, P., Aminghafouri, A., 2018b. Economic evaluation of agroecosystem services of saffron in Khorasan Razavi province. Saffron Agron. Technol. 6 (1), 73
89 (in Persian).

Ecosystem services can be influenced by spatial scale economies (Hein et al., 2006; Kremen, 2005). According to their study, per hectare environmental benefits and costs are much higher on larger farms than on smaller farms. The increase in environmental benefits from these larger saffron farms is much higher than the increase in the environmental costs, so the total net value of environmental benefits increases when farm area is expanded (Fig 23.16). Fig. 23.17 presents the magnitude of each environmental function on different farm sizes of wheat fields in Khorasan Razavi. The difference in economic benefits between large crop farms (10 hectares and more) and small crop farms (below 1 hectare) was estimated by Nassiri and Singh (2009). The biggest difference in environmental benefits between large and small farms is that 40% more oxygen in produced on large farms.

23.6

Green policy analysis matrix of saffron

In this section, we present a “green policy analysis matrix” that includes the value of greenhouse gas emissions and oxygen production. We only include some regulating and producing functions because of data limitations. Saffron yields for the study farms located in Torbat-e Heydarieh in Khorasan Razavi province average 6.25 kg ha21. Using the LCA and assuming that the carbon tax is $14.25 per ton of CO2, the amount of greenhouse emissions and their values are as given in Table 23.11.

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SECTION | V Economy and trade of saffron

FIGURE 23.17 Ecosystem services and negative impacts of wheat fields of various sizes in Khorasan Razavi. From Koocheki, A., Nassiri-Mahallati, M., Aminghafouri, A., Mahloji, M., Fallahpour, F., 2015. Monetary value of agro ecosystem services of wheat fields in Khorasan Razavi province. J. Agroecol. 8, 612
627 (in Persian).

TABLE 23.11 Values of ecosystem services and impacts from saffron production. Gases

Amount per hectarea

Values (dollars)

CO2

995.3125

86.6

CH4

820.625

71.4

N2O

491.25

42.8

Total environmental Impacts

2307.19

200.72

Oxygen

7.5

20.25

Net value




2 180.47

a Data from Nezamoleslami, A., 2018. The Comparative Advantage of Saffron Production in Khorasan Razavi Province with Regard to Greenhouse Gas Emissions. Case Study: Torbat Heydarieh Region (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

TABLE 23.12 Green Policy Analysis Matrix for saffron, per hectare, for a 7-year period. Value

Income

Costs Tradable inputs

Nontradable inputs

Benefit

At market price

65,250

9981.18

28,119

27,149.81267

At shadow price

72,641.75 (72,500)

10,123.39

42,924.046 (41,519.006)

19,594.314 (20,857.604)

Change in shadow price (%)

0.19

0

3.38

2 6.1

DRC (without environmental impacts) 5 0.66

DRC (with environmental impacts) 5 0.69

UC (without environmental impacts) 5 0.732

UC (with environmental impacts) 5 0.71

DRC, Domestic Resource Cost; UC, unit cost. Data from Nezamoleslami, A., 2018. The Comparative Advantage of Saffron Production in Khorasan Razavi Province with Regard to Greenhouse Gas Emissions. Case Study: Torbat Heydarieh Region (M.Sc. thesis). Ferdowsi University of Mashhad, Iran (in Persian).

If the value of oxygen production is added to social revenue and the value of CO2 emissions is added to social domestic inputs, we have the final green policy matrix for saffron (Table 23.12). The values for the PAM elements without ecosystem services and environmental impacts are presented in parentheses for comparison. As the output shows, inclusion of ecosystem services and environmental impacts to the PAM increases the net benefits by 6%. Including environmental impacts significantly changes the PAM analysis for saffron. Domestic resource costs and unit costs also change a little. Khorasan Razavi still has a comparative advantage and competiveness in saffron production.

Environmental economic analysis of saffron production Chapter | 23

23.7

387

Conclusion

This chapter provided an assessment of the economic and environmental aspects of saffron production over its 7-year growth period. The following conclusions are warranted: The highest environmental impacts, including GWP, AP, AEP, and TEP, are mainly generated from cow manure. Saffron’s detrimental impacts are reduced dramatically by properly managing the use of cow manure through strategies involving manure storage, manure composting, and proper time of manure use. The carbon footprint of producing saffron is also reduced if cow manure is properly managed. Saffron is a sustainable and ecofriendly producer of ecological energy. First, a large share of its energy requirements is met by renewable and vast resources such as labor and cow manure. Large amounts of labor are used in saffron production. Cow manure—as the most important energy source of soil fertility—contributes to complete the cycle of nutrition. Organic materials of cow manure improve the dynamic soil property, and thus provide environmental services, in particular provisioning functions, through stimulating biological soil activities. Second, it is possible to manage the use of nonrenewable resources—including fossil fuels and N-based fertilizers—so that their environmental impacts are reduced. The environmental impacts of N-based fertilizer use are dramatically reduced if precise management techniques, such as soil detection methods aimed at improving soil organic materials, are applied. The environmental impacts of fossil fuels are decreased by using biofuels. Additionally, as saffron is commonly grown in arid and semiarid regions where solar energy is abundant, replacing fossil fuels with solar energy would significantly reduce the use of nonrenewable energy sources. Third, saffron has low water requirements and high water use efficiency. Considering the current water crisis and the important role of agricultural water management in resolving it, saffron, as a drought-tolerant crop, can be a good alternative toward sustainable agriculture. Saffron generates considerable economic benefit to farmers. Private income per a hectare of saffron is about six times its costs. Additionally, saffron is labor-intensive farming that promotes local livelihood security, particularly in regions with high unemployment. The economic value of environmental benefits from saffron production is about eight times its environmental costs. The climate and air quality improvement caused by oxygen generation and carbon sequestration is the most important environmental benefit of saffron production. Although the present studies showed a trivial share for the cultural function, observations suggest that public interests in visiting saffron farms have been growing due to saffron festivals. This provides local communities with the potential to increase their income sources and even employment opportunities. Public visits help saffron farms to be preserved and can provide training opportunities to the public. To sum up, saffron production in arid and semiarid regions has a comparative advantage. The advantage is increased even more if environmental services are included.

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209.

Section VI

Saffron and health

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Chapter 24

Saffron in Persian traditional medicine Mahdi Yousefi1 and Khosro Shafaghi2 1

Department of Persian Medicine, School of Persian and Complementary Medicine, Mashhad University of Medical Sciences, Mashhad, Iran,

2

Department of Nutrition and Biochemistry, Faculty of Medicine, Gonabad University of Medical Sciences, Gonabad, Iran

Chapter Outline 24.1 Introduction 24.2 A short history of using saffron in traditional medicine 24.3 A brief survey on principles of Persian medicine focusing on pharmacological aspects 24.3.1 Temperament 24.3.2 Pharmacotherapy 24.3.3 Temperament of drugs and medicines 24.4 Origin and history of the word saffron 24.5 History of saffron usage in Persian civilization

24.1

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24.6 Medical properties of saffron 24.6.1 Botanical aspects 24.6.2 The nature and matter of saffron 24.6.3 Temperament and general properties 24.6.4 Medicinal uses of saffron 24.6.5 Toxicity and adverse effects 24.6.6 Quality assessment 24.7 Conclusion References

397 397 397 398 402 403 403 403 404

Introduction

Iranian traditional medicine (TM) is one of the famous traditional medical systems, which is occasionally called Unani medicine, Arabic medicine, humor medicine, or Islamic medicine. For 30 years now, the World Health Organization (WHO) has supported the development of TM in order to implement the slogan “Health for all by the year AD 2000.” The decision to support TM was based on two foundations: first, lack of access of a great number of people (up to 80% in some countries) to primary healthcare and second, dissatisfaction from the outcomes of treatments by modern medicine, especially in relation to chronic diseases and the side effects of chemical drugs (WHO, 2002). In 2002, the WHO described TM in more detail and used TM as a comprehensive term to refer both to TM systems such as traditional Chinese medicine, Indian Ayurveda, and Arabic-Unani medicine, as well as to various other forms of indigenous medicine. In TM, therapies consist of both medication and nonmedication. Medications in TM include herbal medicines, animal parts, and/or minerals. Nonmedication therapies are carried out primarily without the use of medication as in the cases of acupuncture, manual therapies, and spiritual therapies (WHO, 1978, 2002). In order to use traditional therapeutic methods, understanding and deep insight about principles, fundamentals, and methods is necessary. Thus, a short review on history, principles, and drug terminology in Iranian TM is necessary. The Iran Ministry of Health and Medical Education has replaced the phrase “Iranian traditional medicine” with “Persian medicine,” thus it will be used here. In this chapter, after a brief review of the history and principles of Persian medicine (PM), data on the medicinal uses of saffron, which were obtained from major books on PM, will be discussed. The selected books were the most important sources of medical science and materia medica for more than one thousand years. These resources were searched for information regarding the nature, general properties, therapeutic applications, undesirable effects, and toxicity of saffron.

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00025-3 © 2020 Elsevier Inc. All rights reserved.

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A short history of using saffron in traditional medicine

PM is a medical system with some specific features that may appear very different from conventional medicine. It is necessary that some medical and pharmacological terms be explained before discussion of saffron properties. Saffron has been discussed in traditional literatures for over a thousand years. A short survey of the history of PM is important not only from a historical but also from a medical perspective. The medical history of ancient Persia can be divided into four distinct periods including pre-Islamic, Pahlavi epoch, period of Darius I of Persia, and the medieval Islamic period. The earliest historical records of PM are found in Zend-Avesta, the primary collection of religious texts of Zoroastrianism. The second epoch covers the era of Pahlavi literature, where the entire subject of medicine was systematically treated in an interesting treatise and is claimed to list 4333 diseases. The third era begins with the Achaemenid dynasty and covers the period of Darius I of Persia, whose interest in medicine was said to be so great that he reestablished the school of medicine in Sais, Egypt, which previously had been destroyed, restoring its books and equipment (Benjamin, 1949). In the medieval Islamic period, one of the main roles played by Iranian scholars in science was the conservation, consolidation, coordination, and development of ideas and knowledge in ancient civilizations. Some Iranian physicians such as Muhammad ibn Zakariya ar-Razi (Latinized as Rhazes) and Ibn-Sina (Latinized as Avicenna) accumulated all the existing information on medicine at the time, and added to this knowledge with their own astute observations, experimentation, and skills (Elgood, 1951). “Qanoon fel teb of Avicenna” (“The Canon”) and “Kitab al-Hawi of Razi” (“Continens”) were among the central texts in Western medical education from the 13th to 18th centuries (Osler, 1921; Siraisi, 1987). The practice and study of medicine in Persia has a long and prolific history. The Iranian academic centers like Gundeshapur University (CE 3rd century) were sites for productive interactions between great scientists from different civilizations. These centers successfully followed their predecessors’ theories and greatly extended their scientific research. Persians were the first establishers of the modern hospital system (Behrouz et al., 1993; Meyerhof, 1952; Mohammadali and Shane Tubbs, 2007). PM is a medical system that views the world as good and seeking discipline, created by the wise and omniscient Almighty (Mosaddegh and Naghibi, 2002; Naseri and Shams-Ardakani, 2004). PM consists of the sum total of all the knowledge and practices used in diagnosis, prevention, and elimination of disease in Persia from ancient times to the present. It is based entirely on practical experience and observations passed down from generation to generation. PM originated over 8000 years BCE; some medical historians such as Cyril Elgood believe that PM was more advanced than medicine of Assyria and it is not too bold to go even further. He claimed that Persians taught the principles of Greeks or Unani medicine (Elgood, 1992). After Darius’ murder, countless Persian books and sciences such as medical literature were passed to the Greeks (Abdollah, 1999). Some physicians believe that PM as a holistic system of medicine dates back 14 centuries. According to some historians, PM or Arabic medicine refers to medicine developed in the medieval Islamic civilization and mostly written in Arabic, the lingua franca of the Islamic civilization. Despite this fact, a significant number of scientists during this period were not Arabs. Therefore, the label “Arabic medicine” does not describe the rich diversity of Eastern scholars who have contributed to Islamic science in this era. After the decadence of Greco-Roman medicine, Islamic medicine took over the lead for the subsequent thousand years. Muslims searched for old medical books, read, translated, distributed, and improved upon them (Kamal, 1975). Regardless of the historical origin of PM, currently in the Islamic Republic of Iran, PM is being taught in universities as an academic course and degree and being practiced in clinics by physicians.

24.3 A brief survey on principles of Persian medicine focusing on pharmacological aspects 24.3.1 Temperament According to PM, every person is supposed to have a unique humoral constitution, which represents their healthy state. Any changes in this constitution bring about a change in their state of health. Humans are a part of nature. Nature is constituted by the four elements and the human body by the four humors. Elements and humors have their elementary qualities in common. They form the bridge between the microcosm (individual) and the macrocosm (universe). When the humors are normal in quality and quantity and well mixed so that the condition of eucrasia dominates, a human is healthy. However, as a result of disturbances, one humor can come to dominate in an abnormal way, and the balance will be upset. Thus, the mixture will lose equilibrium, a dyscrasia will occur, and the individual will be sick.

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In these conditions the organism, by its innate healing power called the skillful nature, will try to restore the balance. The humors, which were considered crude in the beginning of the disease, underwent a process of ripening, a coaction, and when they had matured, the faulty matter was driven out in the urine, stools, sputum, or pus. In this way the balance was restored and the patient cured. On the other hand if the disturbance was such that the natural process could not overcome it, the patient succumbed. The very important practical consequence of these views is that the physician is taught to direct his entire treatment in such a way that it would assist the innate healing power of the body and avoid whatever might possibly antagonize it. The humors have elementary qualities and since they determine the character of the diseases, they also have dominating qualities. Drugs, like other objects of nature, have definite qualities (i.e., specific temperaments) and thus a disease that is warm and moist will be cured by drugs that are cold and dry. The theory of four humors can also be used to explain the various constitutional types of humans. No two individuals are the same, and the humoral theory seems to explain these differences (Ibn-Sina, 1987). It was assumed that one of the four humors could slightly dominate physiologically without causing disease. Thus, if black bile (normal cold and dry temperament traditionally called soda) dominated, the individual belonged to the melancholic type. It was the type that many men of genius belonged, including philosophers, statesmen, and artists. There was also a somewhat imbalanced type, which today we could call manic-depressives, the people who sometimes are in high spirits and sometimes deeply depressed. Similarly, it was assumed that blood (traditionally called Dam), phlegm (traditionally called Balgham), and yellow bile (traditionally called Safra) could dominate physiologically and so the physicians described the sanguine, phlegmatic, and choleric types, respectively. Now various branches of medicine, notably immunology, genetics, cytology, hematology, and psychosomatic medicine, have evidence to support humoral theory. These fields have unearthed the truth that, as there are differences in the appearance of features of individuals, there is also a specific temperament with its own individual characteristics.

24.3.2 Pharmacotherapy PM also has a different view on pharmacotherapy. In this field, the PM system possesses a remarkable wealth of knowledge and has made many outstanding contributions to the advancement of medicine. Here, the principles of pharmacology and pharmacotherapy are related to the humoral theory of medicine. The use of a particular drug is governed by three main factors: 1. Nature of the drug 2. Nature of the ailment 3. Temperament of the patient The guiding principle is that the drug to be prescribed should possess qualities opposite to those present in the disease. PM literature on pharmacology contains a detailed view including: 1. 2. 3. 4. 5. 6.

Nature and qualities of drugs Gradation of the potency of drugs (orders) Division of drugs according to quality Action of drugs on various systems or organs of the body Use of purgatives Administration of drugs dealing especially with dosage and timings, modes of Administration, forms and shapes of drugs, correction of harmful effects of drugs, and drug substitutes

PM pharmacology is very much concerned with the classification of drugs. Based qualities, drugs could be classified into four categories: 1. 2. 3. 4.

Drugs with warm temperament Drugs with cold temperament Moist drugs (including lubricants) Dry drugs

However, the most well-known classification is the distinction between simple (traditionally called Mofradat) and compound drugs (traditionally called Morakkabat). Simple drugs occur in their natural and simple state. Compound drugs are formed by mixing two or more simple drugs. The branch of Persian pharmacology dealing with compounds is usually gharabadin, which means drugs formulation (Aqili-Khorasani, 1992).

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24.3.3 Temperament of drugs and medicines PM is based on the theory of temperament. Temperament is a quality (and not matter) resulting from the interaction of opposite properties of elements. This interaction make a uniform quality in all particles. In living creatures four temperaments including hotness, coldness, moistness, and dryness naturally occur (Ibn-Sina, 1987). Each plant has been composed from many components and each component has its’ temperament.The interactions between components of a plant make the final temperament of the plant. They are also assigned temperaments. Drugs are classified by four physical orders. When a drug enters the body, it affects the body’s temperament. A warm drug, after entering the body and interacting with the biological processes, produces (temperamental) effects, which are warm. Drugs are principally prescribed to improve health state and to correct or adjust an abnormal physiological or pathological condition (which could be a result of humoral imbalance), process, or diseasestate of an organ (AqiliKhorasani, 1992). Some definitions are necessary to understand drug terminology in PM.

24.3.3.1 Normal drugs According to Avicenna, when a physician refers to a drug as normal or balanced, they do not mean to state that the drug is in fact normal, nor do they mean that its temperament is completely like that of living beings. They mean that when such drugs interact with the physiological processes of body, they do not create any additional qualities and will not change the temperament.

24.3.3.2 Warm and cold drugs A warm or cold drug produces temperamentally as much warmness or coldness in the body, which is somewhat more than the normal body coldness or warmness. The fingerprint of temperament is exclusive for each individual; therefore, the degree or order of the pharmacological effect of a drug varies per individual but only to a certain extent. Therefore, one drug may be extremely cold or warm for one and only moderately cold or warm for another. For this reason when one drug does not produce desired action it is better to use another drug of the same order.

24.3.3.3 Classification of drugs based on temperament In regards to further classification, each order of drugs has been subclassified into three types: first, second, and third order, or beginning, middle, or end. When a drug is warm in beginning, it means that the action of the drug is weak, while warm in the end indicates potent action of the drug. Thus, drugs belonging to third order in the end shall undoubtedly be toxic in action because these type of drugs affect not only the function but also the structure of an organ and some structural damages may be happened. In order to getting a common understanding about category of drugs’ actions, drugs have been classified into certain orders due to their quick or slow and dose-related (observed or desired) actions. Keeping in mind their pharmacological action and effects, physicians have classified natural drugs into four orders in addition to the normal/balanced order drugs. First-order drugs: The conditions or effects produced by such drugs (i.e., warmness or coldness) are not noticeably perceived by the body or the relevant organ. However, if administered repeatedly, then its actions (general or local) become evident. The action of first-order drugs also becomes noticeable or apparent if they are administered in comparatively large doses. Second-order drugs: The conditions or effects produced by such drugs is comparatively pronounced but not so much as to affect the organs’ function. However, if there already exists an adverse or abnormal condition in the body (like tendency of diarrhea), then it may potentiate or promote such conditions. Desired changes in functions or adverse effects of these drugs may be appeared only in repeated or large dose administration. Third-order drugs: The conditions or effects produced by such drugs due to their action and intensity is apparent or symptomatically detectable, but the damage or harm is not so grave so as to cause fatality or produce acute toxicity. However, their repeated use or administration in large doses is toxic, poisonous, or fatal. Fourth-order drugs (toxic drugs): The conditions or affects produced by such drugs due to their potentially toxic action is mostly harmful or adverse, and their use is advised only in small doses for short durations. Such drugs are either detoxified before use or used with the proper corrigendum. Use of such drugs is advised only in the most serious disease states to evacuate the defined or well identified pathological (mal humor) states that cannot be controlled or treated by drugs of the above three orders. The difference between toxic and poisonous drugs is that the action of toxic

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drugs results in specific toxic conditions (pharmacologically identified states), whereas poisons’ actions proceed due to their molecular structures (which become the cause of adverse chemical changes in the body) (Aqili-Khorasani, 1992).

24.4

Origin and history of the word saffron

The word saffron originated from the Latin word safranum. From the historical and linguistic viewpoint, this word ultimately referred to the term za’far¯an Akkadian azupiranu, “saffron.” In some countries including Catalan, Italy and Portugal, the saffron is named safra`, zafferano, and ac¸afra˜o, respectively, which are derivatives of Latin word safranum (Harper, 2011). In some other languages such as Portuguese and Spanish, the Arabic form az-zafera´n is the source. The Latin term crocus is certainly a Semitic loan word. It is adapted from the Aramaic form kurkema via the Arabic term kurkum and the Greek intermediate κρoκoς krokos, which once again signifies “yellowish” (Kafi et al., 2006; Klein, 1987). Moreover, “the Sanskrit kunkumam might be in some way related to the Semitic term” (Harper, 2011).

24.5

History of saffron usage in Persian civilization

Saffron-based pigments have been found in the prehistoric paints used to illustrate beasts in 50,000-year-old cave art found in modernday Iraq, which was even then northwest of the Persian Empire (Humphries, 1998; Willard, 2002). The Sumerians used saffron as an ingredient in their remedies and magical potions. Sumerians did not cultivate saffron. They gathered their stores from wildflowers, believing that divine intervention alone enables saffron’s medicinal properties (Willard, 2002). Such evidence suggests that saffron was an article of long-distance trade before Crete’s Minoan palace culture reached a peak in the 2nd millennium BCE. In ancient Persia, saffron (Crocus sativus “Hausknechtii”) was cultivated at Derbena and Isfahan in the 10th century BCE. There, Persian saffron threads have been found interwoven into ancient Persian royal carpets and funeral shrouds (Willard, 2002). Saffron was used by ancient Persian worshippers as a ritual offering to their deities and as a brilliant yellow dye, perfume, and medicine. Thus saffron threads would be scattered across beds and mixed into hot teas as a curative for bouts of melancholy. Indeed, Persian saffron threads, used to spice foods and teas, were widely suspected by foreigners of being a drugging agent and an aphrodisiac. These fears grew to forewarn travelers to abstain from eating saffron-laced Persian cuisine (Willard, 2002). In addition, Persian saffron was dissolved in water with sandalwood to use as a body wash after heavy work and perspiration under the hot Persian sun (Willard, 2002). Later, Persian saffron was heavily used by Alexander the Great and his forces during their Asian campaigns. They mixed saffron into teas and dined on saffron rice. Alexander personally used saffron sprinkled in warm bath water, taking after Cyrus the Great. Much like Cyrus, he believed it would heal his many wounds, and his faith in saffron grew with each treatment. He even recommended saffron baths for the ordinary men under him. The Greek soldiers, taken with saffron’s perceived curative properties, continued the practice after they returned to Macedonia (Willard, 2002).

24.6

Medical properties of saffron

24.6.1 Botanical aspects Saffron has been widely used as an herbal medicine, spice, food coloring, and a flavoring agent since ancient times. The leaves of saffron have specific properties. They are narrow and have a filose margin. The flowers of saffron are lily-like. At the base of the flower two bracts are present. Saffron specimens that are usually presented in markets and used in homes are the stigmas of the plant. This filament is 7
11 mm long and bright orange in color (Gruenwald and Brendler, 2007). Cultivation of this plant occurs in many countries including Europe, Turkey, Iran, Central Asia, India, China, and Algeria. The most important region for cultivation of saffron in Iran is the south Khorasan province (Mazhari, 2000).

24.6.2 The nature and matter of saffron Saffron has select properties of taste including slight bitterness, thin astringency, and a very invisible mucilage that makes saffron have a very light sweetness in the mouth. It is concluded that saffron has elements from the soil and water categories of the four elements viewpoint. Saffron is thin (Latif) material, meaning it is capable of dividing into very small particles and penetrates quickly in all compartments of the body. Thus, saffron is suitable for very fine

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sections of body, which is called spirit in PM and could be adapted with energy dimension of body which is discussed in energy based medicine (Gharashi, 2000).

24.6.3 Temperament and general properties Saffron temperament is warm and dry. Most PM literature discusses saffron as follows (Aqili-Khorasani, 1992): 1. A mofarreh agent: A mofarreh drug induces some sensations including vitality, exhilaration, pleasure, delight, fun, and enjoyment. 2. A sense-enforcing agent. 3. Maturative (Monzej): A medicine that moderates humor consistency. 4. Resolvent (Mohalel): Medicines that cut humors from their position, separate their components, and excrete them in vapor form. 5. Modifier (Mosleh): A modifier was defined as a substance that modifies the efficacy of foods and drugs. They were used for improving drug efficacy, reduction of side effects, and masking unpleasant taste or odors. These goals could be achieved in several ways such as synergy, maintaining their effect (prevention from metabolism or stabilization of it in dosage form), sustaining drug release, and convoying drug in whole body or specified organs. Saffron protects Balgham (one of four humors) from infection and degeneration. 6. Diuretic (Moder): A medicine that drives out and excludes water content of foods and body fluids by urination, menstruation, sweeting, and lactation. This is done by hotness and resolvent functions. Saffron help the body to excrete waste material by urination. 7. An astringent (qabez). 8. An agent that increases sexual desire. 9. Tonic (Moghavi): A medicine that moderates organ consistency and temperament. Saffron is tonic for liver, lungs, gastrointestinal tracts, and vital spirit. 10. Antiblockage (Mofateh): Antiblockage medicines mobilize humoral materials and move them out of tracts, pores and organs. Attenuant medicines are drugs that make other substances thinner. Actually, attenuant medicines lower the consistency of humors. This function is not the outcome of coldness because coldness causes compression and contraction of substances and is not for dryness and wetness (passive primary qualities, not active). Therefore, it is only the outcome of hotness. The thinning function of attenuants is helpful in three ways: a. Maturation: Maturation of thick materials is achieved by thinning humors. Additionally, if these substances are viscous, drugs such as vinegar and spicy and sharp substances are useful because they are both attenuant and cutting agents. b. Blockages opening: Thinning of blocking materials facilitates diffusion of them from their locations. The attenuants, which contain detersive and/or abstergent functions, are better antiblockages. c. Resolvent function on thinned substances is easier. Therefore, when resolvent activity is required, attenuants are used. 11. Convoying (Mobadregh): Something that holds the properties of purifying component(s), and also mixes and conveys them to organs, like the function of wine on foods (Yousefi-Heravi, 2008). “Something that is capable to crash another thing, which mixes with it and permeate into (body) organs like water, which do the same with foods and deliver the vital power to them” (Arzani, 2008). Among drug functions, convoying (Badraghe) is unique, mainly due to the relationship of this function with absorption and distribution of other drugs. Most of traditional physicians believe that a convoying plant play the role of a delivery agent. At a glance, the majority of convoys are specific for one or more organs. Most of convoy medicines are hot (82%) and dry (86%). In terms of drug functions, most of convoys were mentioned in literature as antiblockage (82%), diuretic (Moder, 68%), tonic (Moghavi, 64%), resolvent (Mohalel, 59%), attenuant (Molatef, 55%), and abstergent (Monaghi, 46%) (Ebadi et al., 2016). Convoys are considered as a type of modifier in PM. In old authoritative literature, convoys were described as modifiers, which homogenize, mix, and/or guide the components (drug or food) to organs. In addition to these features, facilitation and speeding up characteristics were also emphasized. Therefore, it can be concluded the convoys are substances (or drugs) that facilitate access of drugs and foods to the whole body or organs. Some drug functions attributed to convoys (attenuant, antiblockage, resolvent, diuretic, and cutting activity) originated from the hot primary quality. No indication was found in investigated literature for deciphering the dry primary quality. Among the other functions,

TABLE 24.1 The most important medical aspects of saffron in PM. General medicinal effects

Therapeutic effects

Bitter resolvent

Liver deobstruent

Astringent Bitter Concoctive Disinfectant

Antiinflammatory Aphrodisiac Digestive Diuretic Emetic Exhilarant Gastric tonic Hypnotic Improve complexion Internal organs tonic Liver tonic Oxytocic Pleurisy Respiratory relaxant Respiratory tonic Visual improvement

Astringent Concoctive Resolvent

Hypnotic Improve complexion Internal organs tonic Liver deobstruent Liver tonic Vascular deobstruent

Adverse effectsa

Lethal dosage

References




Tabari (1928)

Headache Hypomania Loss of appetite Harmful for brain Nausea

10.5 g

Razi (1968)

Harmful for stomach Headache Head congestion (Head fullness) Yellow skin




Heravi (1967)

Antilithiasis Antiasthma Conjunctivitis Dropsy Dysentery Eye diseases Gastritis Gastrogenic diarrhea Gout Haemoptysis Hemorrhoids Intestinal excoriation Joints pains Liver diseases Pharyngitis Pharyngitis Rectal collapse Spleen diseases Women genital diseases




Akhawayni-Bukhari (1992)

Astringent Attenuant Concoctive Desiccant Diuretic

Inflammations of internal organs Internal organs tonic Liver deobstruent




Majusi Ahwazi (1877)

Astringent Concoctive Disinfectant Diuretic Resolvent

Antiinflammatory Aphrodisiac Cardiac tonic Deobstruent Emetic

10.5 g

Ibn-Sina (1987)

Headache Hypnotic Dizziness Nausea Loss of appetite Exhilarant Eye diseases Gastric tonic Improve complexion Internal organs tonic Liver tonic Otitis Oxytocic

(Continued )

TABLE 24.1 (Continued) General medicinal effects

Therapeutic effects

Adverse effectsa

Lethal dosage

References

Pleurisy Respiratory tonic Spleen diseases Uterine malignancies Uterine sclerosis Astringent Resolvent Astringent Concoctive Disinfectant Resolvent

Exhilarant Exhilarant Eye diseases Hypnotic Internal organ tonic

Headache dizziness Respiratory relaxant




Jorjani (1976)

Astringent Bitter Concoctive Diuretic Emollient Potent resolvent

Antiinflammatory Aphrodisiac Detoxification of alcohol Emetic Exhilarant

Headache Hypomania Loss of appetite Head fullness Nausea Eye diseases Gastric tonic Hypnotic Improve complexion Internal organ tonic Liver deobstruent Liver tonic Narcotic

10.5 g

Ibn-Beytar (2001)

Concoctive Disinfectant Diuretic Resolvent

Aphrodisiac Cardiac tonic Deobstruent Emetic

Otitis Oxytocic Pleurisy Rectal problems Renal and vesical cleanser Respiratory relaxant Respiratory tonic Uterine diseases Vascular deobstruent Visual improvement Hypnotic Dizziness Nausea Loss of appetite Exhilarant Eye diseases

Astringent Concoctive Diuretic Resolvent

Cardiac tonic Deobstruent Hypnotic Improve complexion Oxytocic Visual improvement

Loss of appetite Headache




Ibn-Nafis Qarshi (2001)

Astringent Resolvent

Antilithiasis Aphrodisiac Arthralgia, gout, and back pains Contraceptive Exhilarant Gastric tonic Hemostatic Liver tonic Oxytocic Palpitation Pharyngitis Pleurisy Stimulant Uterine diseases

Headache Harmful for lungs Loss of appetite

10.5 g

Antaki (2000)

(Continued )

TABLE 24.1 (Continued) General medicinal effects

Therapeutic effects

Adverse effectsa

Lethal dosage

References

Visual improvement Concoctive Diuretic Resolvent of infectious phlegm

Antilithiasis Aphrodisiac Arthralgia Cold headache Erysipelas Exhilarant Eye diseases Gout Hemostatic Hypnotic Improve complexion Induce laughter Internal organ tonic Liver deobstruent Liver tonic Malignancies Otitis Oxytocic Rectal diseases Renal & vesical cleanser Respiratory tonic Spleen deobstruent Uterine diseases Uterus malignancies

Harmful for nerves Headache Nausea Stupor

10.5 g

Tonekaboni (1959)

Astringent Agglutinant Stimulant Treat phlegmatic infections

Antilithiasis Aphrodisiac Arthralgia Brain deobstruent Detoxification of alcohol Eye diseases Gastric tonic Gout Hemostatic Hypnotic Improve complexion Induce laughter Internal organ tonic Liver deobstruent Liver tonic Malignancies Otitis Oxytocic Pleurisy Potent exhilarant Rectal diseases Renal and vesical cleanser Respiratory tonic Severe headache Spleen deobstruent Urinary retention Uterine diseases Uterus malignancies Visual improvement

Dizziness Harmful for kidney Headache Loss of appetite Nausea

10.5 g

Aqili-Khorasani (1992)

a Most of the mentioned side effects, including headache, are observed following consumption of high doses of saffron. Source: From Javadi, B., Sahebkar, A., Emami, S.A., 2013. A survey on saffron in major Islamic traditional medicine books. Iran J. Basic Med. Sic. 16, 1
11.

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attenuant is important in convoys, mainly due to the thinning activity of attenuant function that leads to thin humors and consequently thin organs, which are more diffusible for drugs. In PM, some drug functions are induced or enhanced by the contribution of the other functions (Aqili-Khorasani, 2006; Javadi, 2012). According to the old literature, attenuant function facilitates functions such as antiblockage, maturative, diuretic, and resolvent. Based on PM resources, the amount of convoy used in formulation is often lower than therapeutic doses. A formulator is able to use only the convoying effect by using a lower amount, less than the therapeutic dose, or apply therapeutic effects by using the convoying drug at therapeutic levels (Aqili-Khorasani, 2006; Ebadi et al., 2016; Javadi, 2012). In PM texts, convoy medicines can accompany main drugs to specific organ(s), internal or peripheral compartment(s), and the whole of the body. The affinity of some convoys for specific organs suggests targeting effecst to those organs. On the other hand, the majority of specific convoys have effect(s) on target organ(s), suggesting synergistic effects with the main drug. This phenomenon can be explained by two causes. First, as stated, the dosage for convoying is less than the effective therapeutic dose of the convoy on the target organ. Second, all specific convoys also affect organs other than the target organ(s), but they do not have convoying properties. In theory, it is quite reasonable that there are some pharmacologically active constituents in the herb, which accompany convoying constituent(s) to target organs. Unfortunately, the mechanisms of targeting were not discussed in PM texts. The increase in the rate of delivering the main drug to the target organ and increasing the permeability of the organ to the main drug are probable mechanisms for this effect. In PM saffron is a convoy especially for the heart. It also helps other drugs to penetrate to other organs. According to PM there is special affinity between medicine intended to cure disease and the damaged organ, but the medicine is not able to reach the organ by itself. Therefore, it requires combination with a rapidly penetrating, organ affinitive drug which is convoying agent such as saffron in camphor tablets and heart drugs. Camphor is also used in heart drugs to convoy the cooling effect of itself and other drugs to the heart. Like saffron, camphor weight is less than other drugs in compound heart drugs, as to not overcome other drug properties but to assist and convoy them (Ebadi et al., 2016; Javadi, 2012).

24.6.4 Medicinal uses of saffron Antidepressant properties: One of the most well-known effects of saffron is its exhilarant and antidepressant activity, which leads to the sense of happiness and laughter. Jorjani (1976) noted that “saffron is astringent and resolvent and its fragrance can strengthen these two effects. Hence, its action on enlivening the essence of the spirit and inducing happiness is great.” The use of saffron may also lead to an intense feeling of pleasure, which is close to a psychotic state and may also be used as a sedative (Mollazadeh et al., 2015; Razi, 1968). Treatment of ocular disorders: Saffron was traditionally used to prepare a special eye formulation called collyrium (Kohl) to treat a range of ophthalmic disorders such as painful eye, purulent eye infection cataract, and conjunctivitis and to improve vision. According to Razi (1968) it stops excessive discharges of the eyes when applied with human milk. Treatment of respiratory disorders: Saffron has been traditionally prescribed to improve respiratory function, asthmatic problems, and as a lung tonic. Cardioprotective effects: Saffron is a heart tonic (cardiotonic and cardioprotective) that has been used to support cardiovascular functions and to treat palpitations. Saffron can improve blood flow and nutrition to the heart. Saffron also has antithrombotic and thrombolytic activities (Javadi et al., 2013; Mollazadeh et al., 2015) Gastro-hepatoprotective effects: This plant is a powerful liver tonic and hepatic deobstruent. Tabari described the hepatoprotective effects of saffron stating, “It is warm, moderate, and dry. It is resolvent and bitter. Therefore, it can treat liver obstructions.” Saffron is a gastric tonic and suppresses the appetite. Razi notes that “saffron is a digestive drug with astringent properties. It cleanses the stomach.” Morever, “saffron neutralizes gastric acid, cleanses the stomach, increases digestion of food, strengthens liver and stomach and decreases appetite” (Javadi et al., 2013; Mollazadeh et al., 2015). Oxytocic properties: One of the most important effects of saffron is its potent oxytocic activity, which is exerted even after vaginal use. Hence, the plant has traditionally been prescribed to facilitate difficult labors. According to Razi (1968), “ingestion of two derhams of saffron induces the delivery. I prescribed it many times and the results were always positive. It was useful for the treatment of female genitourinary system disorders. It is prescribed in hardness, blockage, adhesions, and malignant ulcers of the uterus.” Derham is a unit of weight formerly used by Persian traditional physicians. It is difficult to estimate comparative exchange rates with modern weight units, but some scholars state that each derham is equivalent to 3.4 g. Drachma is also a unit of weight mostly used in ancient Greece. The weight of the silver drachma was approximately 4.3 g or 0.15 oz. Some other traditional physicians believed that the

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maximum oral dose of saffron is two Derhams and that more than three Derhams is lethal. Ingestion of one mesqal (another ancient unit of weight estimated to be equivalent to 4.5 g) as a single dose may accelerate labor and delivery of the placenta. Saffron has also contraceptive effects and abortion is one of the side effects of overdose (AqiliKhorasani, 1992). Saffron as a labor facilitator in doses of more than 5 g promotes potent oxytocic activity and this side effect has been used in some cases to induce delivery. Antaki notes that, “it has been experienced that oral use of 3.5 g saffron with rose water and sugar can facilitate delivery. Application of a vaginal suppository prepared by 3.5 g of saffron accelerates labor and delivery of the placenta. It has also contraceptive effects” (Javadi et al., 2013; Mollazadeh et al., 2015; Razi, 1968). Treatment of urogenital disorders: This plant has also been reported to be useful for the treatment of female genitourinary system disorders such as premenstrual syndrome, dysmenorrhea, and irregular menstruation. In PM, it was used as a diuretic and as a purifier of kidney and bladder. It also was used to treat urinary obstruction (Javadi et al., 2013; Mollazadeh et al., 2015; Razi, 1968). Aphrodisiac properties: Saffron also possesses aphrodisiac properties and hence was used to cure impotence. Saffron could improve sexual behaviors include increasing of libido, enhancement of erectile function, and improvement of semen quality. Razi reported that “it is a diuretic and a stimulant of sexual desire” (Javadi et al., 2013; Mollazadeh et al., 2015; Razi, 1968). Anticancer effects: The effects of saffron in the treatment of tumors and malignancies, in particular uterus malignancies, have been mentioned in the Canon of Medicine and other related texts. Ibn-Sina noted that, “local application of saffron with beeswax or egg yolk and olive oil is effective to treat uterus malignancies” (Aqili-Khorasani, 1992; IbnSina, 1987; Javadi et al., 2013; Mollazadeh et al., 2015; Razi, 1968; Tonekaboni, 1959). Cosmetic effects: Saffron is a good drug for some skin diseases. It can be used to cure acne, to refresh facial skin, and to make the skin look more youthful and brighter (Razi, 1968). Antiinflammatory and antinociceptive effects: Razi states that, “saffron features include: softening and quenching boils, improving internal organ pain. Rectal suppository form and ointment of saffron are utilized in the pain of the uterus and anus. It is also painted on erysipelas and is useful in hot swellings of the ear. The smell of its oil reduces inflammation of the liver and heart” (Mollazadeh et al., 2015; Razi, 1968).

24.6.5 Toxicity and adverse effects The most frequent adverse effects of saffron mentioned in studied books were headache, nausea, head fullness, dizziness, hypomania, and appetite suppression. Regarding the undesirable effects of saffron, Aqili-Khorasani (1992) stated that, “it can cause headache and its consumption with wine results in intoxication. Long-term use of saffron can lead to dizziness and damage to nervous system. Aniseed and oxymel can correct these adverse effects.” Skin yellowing is another side effect reported for saffron (Heravi, 1967). Razi also notes that, “three mithqal (13.5 gm) of saffron makes a man so overjoyed that, as a result, high consumption of saffron does not have a good effect on the brain and may be fatal. Abuse of it might cause insane behavior” (Mollazadeh et al., 2015; Razi, 1968). The accumulation of saffron in sclera, skin, or mucosa can produce yellowish visage and mimics icteric complaints (Javadi et al., 2013; Mollazadeh et al., 2015; Razi, 1968).

24.6.6 Quality assessment Ibn-Sina (1987) described high-quality saffron as follows: “Fresh saffron of high quality is characterized by nice color and fragrance. The upper parts of its stigma should be whitish in color. Saffron should not be moldy. It should be neither too compact and thick nor crumbling. Besides, it should not easily impart its color on touch.” Temperament, medicinal and adverse effects, and lethal dosage of saffron in primary PM books are listed in Table 24.1 (Javadi et al., 2013).

24.7

Conclusion

Saffron is one of the most frequently used herbs in PM. The temperament of saffron is hot and dry based on Persian medical literature. Saffron has bitter, stimulant, fragrant, tonic, and aphrodisiac properties. It also has oxytocic, anticarcinogenic, stomachic and antispasmodic, emmenagogue, diuretic, exhilarant, antidepressant, laxative, galactagogue, and antiasthma effects. One the most important biological actions of saffron is to convoy, meaning saffron can increase the bioavailability and absorption of other drugs, especially to the heart.

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References Abdollah, D.A., 1999. Luminaries of Physics in Islamic Civilization, Quoted From Ibn Khaldoon. Darolfekr Publications, Tehran (in Persian). Akhawayni-Bukhari, A.B., 1992. Hedayat Al-Mota’allemin Fi Al-Tibb (An Educational Guide for Medical Students). Ferdowsi University of Mashhad Publication, Mashhad (in Persian). Antaki, D., 2000. Tazkere Oulol-albab (Memorandum Book). Dar-al-Kotob al-ilmiyah, Beirut, Labenon. Aqili-Khorasani, S.M.H., 1992. Makhzan al-Adwiah (Drug Treasure). Enqelab-e Eslami Publishing and Educational Organization, Tehran (in Persian). Aqili-Khorasani, S.M.H., 2006. Kholasat Al-Hekmah. Esmaili Publication, Qom (in Persian). Arzani, A., 2008. Teb-e-Akbari. Jalaledin Publication, Qom (in Persian). Behrouz, R., Ourmazdi, M., Reza’i, P., 1993. Iran
the Cradle of Science. Almanac, Tehran. Benjamin, L.G., 1949. Medicine Throughout Antiquity. F.A. Davis Co, India. Ebadi, N., Masoomi, F., Yakhchali, M., Sadati Lamardi, N., Shams-Ardakani, M.R., Sadeghpour, O., 2016. Convoy drugs in traditional Persian medicine: the historical concepts of bioavailability and targeting. Trad. Integr. Med. 1, 18
27. Elgood, C., 1951. A Medical History of Persia and the Eastern Caliphate From the Earliest Times to the Year AD 1932. Cambridge University Press, London. Elgood, C., 1992. Iran Medical History and the Regions of Eastern Caliphate. Amirkabir Publication, Tehran. Gharashi, A., 2000. Al-Shamel Fi Al-Sanaat Al-Tabeat. Research Institute for Islamic and Complementary Medicine, Tehran (in Persian). Gruenwald, J., Brendler, T., 2007. PDR for Herbal Medicines. Thomson Healthcare Inc, Montvale, NJ. Harper, D., 2011. Online Etymology Dictionary. Available from: ,https://www.etymonline.com.. Heravi, M., 1967. Al-Abniyah and Haqayeq Al-Adwiyah (Basics of Realities on Drugs). University of Tehran Publications, Tehran (in Persian). Humphries, J., 1998. The Essential Saffron Companion. Ten Speed Press, Berkeley, CA. Ibn-Beytar, Z., 2001. Al-Jamee Le-Mofradaat Al-Adwiah wal-Aghziyah (Comprehensive Book in Simple Drugs and Foods). Dar- Al-Kotob Alilmiyah, Beirut. Ibn-Nafis Qarshi, A.D., 2001. Al-Mujaz Fi’l-Tibb (A Commentary on Ibn-Sina’s Canon). Ihyaa al-Torath al-Islami, Cairo. Ibn-Sina, A., 1987. Al-Qanun Fi’l-Tibb (Canon of Medicine). I.H.M.M.R. Printing Press, New Delhi. Javadi, B., 2012. Analytical Research and Compilation of the Basics of Islamic Traditional Pharmacy and Preparation of a Dosage Form Based on Ibn Nafis Books (Ph.D. thesis). School of Traditional Medicine, Tehran University of Medical Sciences, Tehran (in Persian). Javadi, B., Sahebkar, A., Emami, S.A., 2013. A survey on saffron in major Islamic traditional medicine books, Iran J. Basic Med. Sic., 16. pp. 1
11. Jorjani, S.E., 1976. Zakhireh Kharazmshahi (Treasure of Kharazmshahi). The Iranian Culture Foundation, Tehran (in Persian). Kafi, M., Koocheki, A., Rashed, M.H., Nassiri, M., 2006. Saffron (Crocus sativus) Production and Processing. Science Publishers, Tehran. Kamal, H., 1975. Encyclopedia of Islamic Medicine. General Egyptian Book Organization, Cairo, Egypt. Klein, E., 1987. A Comprehensive Etymological Dictionary of the Hebrew Language for Readers of English. Carta, Jerusalem. Majusi Ahwazi, A., 1877. Kamel-al-Sanaat Al-Tibbiah (The Perfect Art of Medicine). Al-Matbaah al-Misryyah, Bulaq. Mazhari, N., 2000. Iridaceae. Research Institute of Forests and Rangelands, Tehran (in Persian). Meyerhof, M., 1952. Science and Medicine. Oxford University Press, London. Mohammadali, M.S., Shane Tubbs, R., 2007. The history of anatomy in Persia. J. Anat. 359
378. Mollazadeh, H., Emami, S.A., Hosseinzadeh, H., 2015. Razi’s Al-Hawi and saffron (Crocus sativus): a review. Iran J. Basic Med. Sci. 18, 1153
1166. Mosaddegh, M., Naghibi, F., 2002. Iran’s traditional medicine, past and present. TMRC 2
20. Naseri, M., Shams-Ardakani, M.R., 2004. The School of Traditional Iranian Medicine: the definition, origin and advantages. JISHIM 17
21. Osler, W., 1921. The Evolution of Modern Science. Yale University Press, New Haven. Razi, M.Z., 1968. Al-Hawi Fi’l-Tibb (Comprehensive Book of Medicine). Osmania Oriental Publications Bureau, Hyderabad. Siraisi, N.G., 1987. Avicenna in Renaissance Italy: the Canon and Medical Teaching in Italian Universities After 1500. Princeton University Press, Princeton, NJ. Tabari, A., 1928. Ferdows Al-Hekmah Fi Al-Tibb (Paradise of Wisdom on Medicine). Aftab Press, Berlin (in Persian). Tonekaboni, M.M., 1959. Tohfat Al-Momenin (Rarity of the Faithful). Mostafavi Press, Tehran (in Persian). Willard, P., 2002. Secrets of Saffron: The Vagabond Life of the World’s Most Seductive Spice. Beacon Press, Boston, MA. World Health Organization, 1978. The Promotion and Development of Traditional Medicine-Report of WHO Meeting. WHO Report Series, Switzerland. World Health Organization, 2002. WHO Traditional Medicine Strategy 2002
2005. Geneva. Yousefi-Heravi, M., 2008. Bahr Al-Javaher. Jalaledin Publication, Qom (in Persian).

Chapter 25

Antiinflammatory and immunomodulatory effects of saffron and its derivatives Mohammad-Hossein Boskabady1,2, Zahra Gholamnezhad1,2, Mohammad-Reza Khazdair3 and Jalil Tavakol-Afshari4 1

Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 2Department of Physiology, School of

Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 3Cardiovascular Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran, 4Immunology Research group, Buali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 25.1 Introduction 25.2 Antiinflammatory effects of saffron and its derivatives 25.2.1 Antiinflammatory effects of saffron extracts 25.2.2 Antiinflammatory effects of saffron petals 25.2.3 Antiinflammatory effects of saffron derivatives 25.3 Immunomodulatory effects of saffron and its derivatives

25.1

405 406 406 407 409 412

25.3.1 Immunomodulatory effects of saffron extracts 25.3.2 Immunomodulatory effects of saffron derivatives 25.4 Conclusion References Further reading

414 415 417 417 421

Introduction

Saffron, or Crocus sativus L., is one of the most widely-used medicinal plants. It was traditionally used for dietary purposes as a spice or food additive, as well as for medicinal purposes. The stigma of saffron is the main part of the plant, and it has therapeutic effects which are the focus of biomedical research (Caballero-Ortega et al., 2007). Chemical analysis of saffron stigmas has demonstrated the presence of volatile and nonvolatile compounds such as proteins, amino acids, carbohydrates vitamins, minerals, and pigments. The volatile compounds contain more than 34 ingredients, including terpenes and their esters. Among these, safranal is the major ingredient. Chemical analysis of the plant extracts has demonstrated the presence of carotenoids, namely crocin, crocetin, picrocrocin, and safranal, which are the main derivatives of this plant (Caballero-Ortega et al., 2007). In addition to the effects produced by saffron stigmas, the flower of the plant possesses some medical effects as well. Saffron is used as a sedative, antispasmodic, aphrodisiac, appetizer, carminative, flatulence, expectorant, sedative, expectorant, rejuvenator, diaphoretic, tranquilizer, eupeptic, and abortifacient in traditional medicine (Abdullaev and Espinosa-Aguirre, 2004; Rı´os et al., 1996). Saffron is also useful in various pharmacological activities as an antidiabetic (Kianbakht and Mozaffari, 2009), anticancer (Tseng et al., 1995), immunomodulator (Boskabady et al., 2011), analgesic (Hosseinzadeh and Younesi, 2002), antimicrobial (Yousefi et al., 2014), antiatherogenic (Xu et al., 2005), cardioprotective (Zhang et al., 2009), antioxidant (El-Beshbishy et al., 2012), and antiinflammatory agent (Hassan et al., 2015). When used as a smooth muscle relaxant (Boskabady and Aslani, 2006), saffron, as well as its derivatives, has a stimulatory effect on β2-adrenoceptors of tracheal smooth muscle (Nemati et al., 2008), an inhibitory effect on the calcium channel of heart (Boskabady et al., 2008), and an inhibitory effect on histamine H1 receptors of tracheal smooth muscle (Boskabady et al., 2008). Additionally, other pharmacological uses of the saffron plant include its use as a hepatoprotective (Sun et al., 2014), renal protective (Hazman and Bozkurt, 2015), gastro-protective (El-Maraghy et al., 2015), Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00026-5 © 2020 Elsevier Inc. All rights reserved.

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antithrombotic (Yang et al., 2008), antimutagenic (Bhandari, 2015), antigenotoxic (Premkumar et al., 2006), and tumoricidal agent (Wang et al., 1995). Saffron and its derivatives produce antiinflammatory and immununoregulatory effects by modulating proinflammatory cytokines and immune factors (El-Beshbishy et al., 2012; Hassan et al., 2015). As this occurs, the inflammatory process and the immune system are stimulated by physical, chemical, and biological factors (Gholamnezhad et al., 2015), causing interactions between the inflammatory process and the immune system in the presence of various inflammatory disorders (Balkissoon et al., 2011; Domej et al., 2014). Inflammation has been shown to play a key role in the pathogenesis of diseases and disorders such as allergy, asthma, and cardiovascular disorders (Aggarwal and Harikumar, 2009). Therefore finding a natural therapeutic agent for preventing and treating inflammatory disease is the aim of many studies (Gholamnezhad et al., 2015). In this chapter, the antiinflammatory and immunomodulatory effects of saffron and its derivatives (based on in vitro and in vivo studies) will be reviewed.

25.2

Antiinflammatory effects of saffron and its derivatives

Inflammation is the root of numerous diseases, thus, discovering new preventive and multipotential agents for effective treatment of inflammatory processes has been of great interest. The antiinflammatory effects of saffron extracts and derivatives have been indicated in several studies, studies which are reviewed in this chapter. Inflammatory mediators such as eicosanoids, oxidants, cytokines, and lytic enzymes are secreted by macrophages, neutrophils, and inflammatory cells (Gholamnezhad et al., 2015). Platelets also play an important role in allergic inflammation; platelet activation has been involved in different inflammatory lung diseases by producing several inflammatory mediators (Averill et al., 1992; Kowal et al., 2006). As these mediators are produced, inflammation is amplified by local responses of the epithelium, smooth muscle, and fibroblast cells through the production of chemokines, cytokines, and proteases. As an example, the main characteristic of asthma is increased airway responsiveness or airway hyperresponsiveness, which is due to lung inflammation (Cohn et al., 2004).

25.2.1 Antiinflammatory effects of saffron extracts In confirming the effect of saffron extracts on airway inflammation, hydroethanolic extract of saffron extracts (0.1, 0.2, and 0.4 mg mL21) were tested on ovalbumin (OVA)-sensitized guinea pigs significantly reduced serum levels of endothelin-1 (ET-1) and total protein (TP) compared to untreated sensitized group (Gholamnezhad et al., 2013). The results indicate that treatment of sensitized animals with dexamethasone and saffron extract reduced serum levels of ET-1 and TP when compared to untreated animals. The effects of 0.4 mg mL21 of the extract were significantly higher than those of dexamethasone (50 μg mL21). Increased serum TP in an asthmatic patient may be due to increased γ globulin, C reactive protein, and other proteins structured as inflammatory mediators (Niimi et al., 1998). The effects of the saffron extract and dexamethasone treatment on lung inflammation were also examined in an animal model of allergic asthma. Treatment of OVA-sensitized animals with saffron hydroethanolic extract (0.1, 0.2, and 0.4 mg mL21) prevented the increase in total white blood cells (WBCs), eosinophil, and lymphocyte numbers. In addition, the effect of the extract on the reduction of WBC count was similar to that of dexamethasone. However, the effect of dexamethasone treatment was less than the effect of low concentration of saffron extract (0.1 mg mL21) on eosinophil count (Bayrami and Boskabady, 2012; Boskabady et al., 2012). The effects of saffron hydroethanolic extract on pathological disorders in the lungs of sensitized animals include improvements to interstitial eosinophil and lymphocyte infiltration, interstitial cell infiltration, atelectasis, lung congestion, bleeding, and epithelial damage. Treatment of sensitized guinea pigs with the plant extract significantly ameliorated pathological indices in the lung. This data also suggested a preventive effect of saffron extract on lung inflammation of sensitized animals (Boskabady et al., 2012). Hydroethanolic extract of C. sativus (50, 100, and 200 mg kg21) on sensitized rats reduced the WBC number and decreased the percentage of neutrophils and eosinophils in the lung lavage as compared to the untreated sensitized animals (Mahmoudabady et al., 2013). In a similar study, saffron hydroethanolic extract also significantly decreased WBC count, eosinophil and neutrophil percentages, RBC and platelet count in the blood of sensitized rats. Lymphocyte percentage was increased in the animals receiving 100 mg kg21 of saffron hydroethanolic extract (Vosooghi et al., 2013). The results of this study indicated the preventive effect of the plant on airway inflammation in sensitized animals, which may indicate the therapeutic effect of the plant on allergic asthma. The reduction of eosinophil, neutrophil and lymphocyte counts in sensitized animals treated with saffron extract suggests that the extract has antiinflammatory properties. In addition, decreases in RBC and platelet counts in sensitized animals treated with saffron extract indicated that the extract may be a useful treatment for different inflammatory lung

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TABLE 25.1 Antiinflammatory effects of various extracts of saffron. Extract

Effect

Experimental model

References

HEE

Reduced serum levels of endothelin-1 and total protein

Sensitized guinea pigs

Gholamnezhad et al. (2013)

HEE

Reduced total WBC, eosinophil and lymphocyte counts in blood and lung lavage

Bayrami and Boskabady (2012)

HEE

Ameliorated pathological lung indices

Boskabady et al. (2012)

HEE

Decreased tracheal responsiveness to both methacholine and OVA and serum levels of inflammatory mediators

Byrami et al. (2013)

HEE

Reduced WBC cells and decreased the percentage of neutrophils and eosinophils in lung lavage

Sensitized rats

Mahmoudabady et al. (2013)

EE

Reduction of proinflammatory mediators (TNF-α, IL-6, and IL-1β)

Chronic constriction injury in rats

Amin et al. (2014)

Reduced ear edema and showed antinociceptive effects

Acetic acid-induced writhing in mice

Hosseinzadeh and Younesi (2002)

Ameliorated paw edema

Formalin-induced paw edema

Hosseinzadeh and Younesi (2002)

AE EE AE of stigma EE AE of petal

AE, Aqueous extract; EE, ethanol extract; HEE, hydroethanolic extract; IL, interleukin; OVA, ovalbumin; TNF-α, tumor necrosis factor-α.

diseases, among which is asthma. The hydroethanolic extract of saffron (20, 40, and 80 mg kg21 day21) significantly decreased tracheal responsiveness (tracheal responsiveness is a key predictor of future obstructive respiratory disease, especially asthma) to methacholine, OVA, and serum levels of inflammatory mediators when compared to untreated sensitized animals. The effects of the highest concentration of the saffron extract (80 mg kg21) were greater than those of dexamethasone (10 mg kg21). These findings indicated that the extract of the plant could attenuate serum levels of inflammatory mediators as well as decreased tracheal responsiveness to methacholine and OVA (Byrami et al., 2013). In confirming the effects on nonairway inflammation, it was observed that ethanol and aqueous saffron extracts (200 mg kg21, i.p.) reduced neuropathic pain in the chronic constriction injury model through reduction of proinflammatory factors (Amin et al., 2014). The antinociceptive and antiinflammatory activities of aqueous (0.8 g kg21; i.p.) and ethanol extracts of saffron stigma (0.1, 0.2, and 0.4 g kg21; i.p.) and petals (0.4, 0.8, and 1.6 g kg21; i.p.) in mice were evaluated using the hot-plate and writhing tests as antinociceptive activity indices. Results of this evaluation showed that extracts have antinociceptive activity against acetic acid-induced writhing as well as weak to moderate activity against acute inflammation using xylene-induced ear edema in mice. In chronic inflammation, both aqueous and ethanol stigma extracts, as well as ethanol petal extract, showed antiinflammatory effects in formalin-induced paw edema in rats. The aqueous and ethanol extracts of saffron stigma and petal showed antinociceptive activity as well as acute and/or chronic antiinflammatory activity (Hosseinzadeh and Younesi, 2002). The antiinflammatory effects of saffron are summarized in Table 25.1.

25.2.2 Antiinflammatory effects of saffron petals 25.2.2.1 Kaempferol In confirming the effect of fresh saffron flower petals on airway-inflammation, kaempferol, a polyphenolic compound, was isolated from the petals (Hadizadeh et al., 2010). The effects of kaempferol on epithelial-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1) showed that kaempferol (10, 25, and 50 μM) significantly blocked the increased cell migration by TGF-β1 induced EMT in human nonsmall lung cancer cells (A549). To do this, the kaempferol recovers the loss of E-cadherin and blocks the induction of mesenchymal markers as well as the upregulation of TGF-β1 mediated matrix metalloproteinase-2 (MMP-2) activity. Furthermore, activation of a kinase (Akt1) was required for TGF-β1-mediated induction of EMT, cell migration, direct phosphorylation of

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a protein (Smad3) at Thr179, and elimination of TGF-β1-induced Akt1 phosphorylation. These results indicated that kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1, which mediated phosphorylation of Smad3 at Thr179 residue (Jo et al., 2015). Lipopolysaccharide (LPS)-induced lung injury in BALB/c mice showed overproduction of proinflammatory cytokines in bronchoalveolar lavage fluid (BALF), including TNF-α, IL-1β, and IL-6, in which kaempferol (100 mg kg21, i.g.) strongly reduced the cytokines. Kaempferol also significantly inhibited LPS-induced alveolar wall thickness, leukocyte infiltration and alveolar hemorrhage in lung tissue with evidence of reduced myeloperoxidase (MPO) activity and increased superoxide dismutase (SOD) activity. Additionally, kaempferol significantly blocked the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways induced by LPS. These results suggested that kaempferol exhibits a protective effect on LPS-induced acute lung injury by suppressing MAPKs and NF-κB signaling pathways and is involved in the inhibition of oxidative injury and inflammatory processes (Chen et al., 2012). The effects of kaempferol (1
20 μM) on inflammation in human airway epithelial cells (BEAS-2B) showed that kaempferol inhibited the expression of toll-like receptor 4 (TLR4), a promotor of inflammatory mechanisms which is significantly increased by LPS. Kaempferol also reduced cellular expression of IL-8 through stimulation (Gong et al., 2013). The levels of C-C chemokine receptor type 3 (CCR3) and eotaxin-1 protein in the lung tissue were enhanced in OVA-exposed mice but the supplementation of the kaempferol (10 and 20 mg kg21, p.o.) dose dependently eliminated the production levels of CCR3 and eotaxin-1. OVA exposure also increased macrophage inflammatory protein 2 (MIP2) and C-X-C chemokine receptor type 2 (CXCR2) productions in mouse lung tissue, in which kaempferol supplementation markedly reduced MIP-2 and CXCR2 production (Gong et al., 2013). Kaempferol could be capable of modulating allergic airway disease either as a preventive (administered 1 hour before OVA sensitization) or curative (OVA sensitization at days 18
21) treatment in sensitized mice models (Medeiros et al., 2009). Kaempferol (3, 30, or 100 mg kg21, s.c.) also reduced the total leukocyte and eosinophil counts similar to the effect of dexamethasone (1 mg kg21) in the BALF (Medeiros et al., 2009). Additionally, kaempferol has effects on other- types of inflammation as well. Kaempferol (20, 40, 60, 80, and 100 μM) suppressed mRNA expression of MMP-2 to restrain the migration of oral cancer cells by inhibiting the c-Jun pathway and extracellular signalregulated protein kinases 1 and 2 (ERK1/2) phosphorylation in a dose-dependent manner (Lin et al., 2013). Furthermore, kaempferol (5
20 μM) significantly reduces vascular endothelial growth factor (VEGF) gene expression at mRNA and protein levels, and significantly inhibited angiogenesis and tumor growth in the cancer cells. Kaempferol treatment also downregulated HIF-1α (a regulator of VEGF) in ovarian cancer cells in a dose-dependent manner (Luo et al., 2009). Kaempferol (20 μM) inhibited secretion of β-hexosaminidase and histamine and reduced the production and mRNA expression of inflammatory cytokines (IL-4 and TNF-α) in immunoglobulin E (IgE)-sensitized (RBL-2H3) cells. Kaempferol also inhibited (IgE)-mediated phosphorylation of phospholipase Cγ, protein kinase C (PKC)μ, and the MAPKs: extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (Kim et al., 2014b). The effects of kaempferol on the IL1β-induced proliferation of rheumatoid arthritis synovial fibroblasts (RASFs) and the production of MMPs, cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) showed that kaempferol (100 μM) inhibited the proliferation of unstimulated and IL1β-stimulated RASFs, as well as the mRNA and protein expression of MMP-1, MMP3, COX-2, and PGE2 induced by IL1β. Kaempferol inhibited the activation of NFκB induced by IL1β and also inhibited the phosphorylation of ERK1/2, p38 and JNK (Yoon et al., 2013). Moreover, kaempferol (30 μM) significantly decreased the mRNA expression of TNF-α in LPS-activated J774.2 macrophages. IL1β gene expression in LPSinduced J774.2 macrophages were also inhibited by kaempferol (Kowalski et al., 2005). LPS (100 ng mL21) enhanced induced nitric oxide synthase (iNOS) mRNA expression, NF-κB activity and signal transduction and activator of transcription 1 (STAT-1), which are important transcription factors for iNOS. Kaempferol (10
100 μM) considerably inhibited iNOS protein and mRNA expression as well as nitric oxide (NO) production. Kaempferol also inhibited the activation of NF-κB and STAT-1 in a dose-dependent manner in J774 macrophages (Ha¨ma¨la¨inen et al., 2007). Treatment of diabetic mice with kaempferol (25, 50, and 100 mg kg21, p.o.) attenuated the development of diabetic neuropathy and reduced pain sensation. Furthermore, kaempferol reduced IL-1β, TNF-α, lipid peroxidation, and nitrite (Abo-Salem, 2014). Studies demonstrated that kaempferol (30 and 150 mg kg21, p.o.) decreased the levels of TNF-ɑ and IL-1β in the serum of high cholesterol-fed rabbits. In addition, kaempferol downregulated mRNA and protein expression of inflammatory molecules such as E-selectin (E-sel), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and macrophage chemoattractant protein-1 (MCP-1) in the aorta of rabbits (Kong et al., 2013). Kaempferol (50 mg kg21, p.o.) markedly inhibited the antigen-induced passive cutaneous anaphylaxis (PCA) response in IgE-sensitized mice (Kim et al., 2014b). It has been reported that kaempferol (2 or 4 mg kg21 day21, p.o.)

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TABLE 25.2 Antiinflammatory effects of kaempferol, a constituent of saffron petal. Effect

Experimental model

References

Blocked the cell migration, upregulation of MMP-2 activity, and eliminated TGF-β1-induced Akt1 phosphorylation

Lung cancer cells

Jo et al. (2015)

Inhibited the expression of TLR4 and decreased cellular expression of IL-8

BEAS-2B cells

Gong et al. (2013)

Reduced TNF-α, IL-1β, and IL-6, inhibited wall thickness, leukocytes infiltration, and alveolar hemorrhage in lung tissue. In addition, significantly blocked the activation of MAPKs and NF-κB signaling pathways.

LPS-induced BALB/c mice

Chen et al. (2012)

Eliminated levels of CCR3 and eotaxin-1 and reduced MIP-2 and CXCR2 production

OVA-exposed mice

Gong et al. (2013)

Reduced total leukocyte and eosinophil counts

OVA-sensitized mice

Medeiros et al. (2009)

Suppressed mRNA expression of MMP-2

Oral cancer cells

Lin et al. (2013)

Reduced VEGF gene expression at mRNA and protein levels and significantly inhibited angiogenesis and downregulation of HIF-1α

Ovarian cancer cell

Luo et al. (2009)

Inhibited secretion of β-hexosaminidase and histamine and reduced the production of inflammatory cytokines

RBL-2H3 cells

Kim et al. (2014b)

Kaempferol inhibited the activation of NFκB and phosphorylation of ERK1/2, p38 and JNK

RASFs cells

Yoon et al. (2013)

Inhibited iNOS protein and mRNA expression as well as NO production and inhibited the activation of NF-κB and STAT-1

J774 macrophages

Ha¨ma¨la¨inen et al. (2007)

Attenuated the development of diabetic neuropathy, reduced pain sensation, and reduced IL-1β, TNF-α, lipid peroxidation, and nitrite

Diabetic mice

Abo-Salem (2014)

Downregulated mRNA and protein expression of E-selectin, ICAM-1, VCAM-1, and MCP-1

High cholesterol-fed rabbits

Kong et al. (2013)

Inhibited the antigen-induced passive PCA

IgE-sensitized mice

Kim et al. (2014b)

Inhibited NF-κb function (NIK)/iκb kinase (IKK), and MAPK signal pathways

Aged rat

Park et al. (2009)

Akt1, Activation a kinase; CCR3, C-C chemokine receptor type 3; CXCR2, C-X-C chemokine receptor type 2; ERK, extracellular signal
regulated kinases; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinases; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases; MCP-1, monocyte chemoattractant protein-1; MIP-2, macrophage inflammatory protein 2; MMPs, matrix metalloproteinase; NIK, nuclear factor-inducing kinase; NF-κB, nuclear factor-kappa B; OVA, ovalbumin; STAT, signal transducer and activator of transcription; TGF-β1, transforming growth factor-β1; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesionmolecule-1; VEGF, vascular endothelial growth factor.

could inhibit NF-κB function by inhibiting the activation of nuclear factor-inducing kinase (NIK)/IκB kinase (IKK) and MAPKs signal pathways in aged rat kidneys (Park et al., 2009). Therefore kaempferol plays antiinflammatory roles by modulating the gene and protein expression of inflammatory molecules. The antiinflammatory effects of saffron petals are summarized in Table 25.2.

25.2.3 Antiinflammatory effects of saffron derivatives 25.2.3.1 Crocin In studying the effect of crocin, a saffron derivative, on airway-inflammation, the effects on hyperreactivity in OVAsensitized mice were observed. Results indicated that crocin significantly suppressed airway inflammation and hyperreactivity. In addition, crocin decreased the levels of inflammatory cytokines, tryptase, and eosinophil peroxidase in lung lavage. It also inhibited the expression of lung eotaxin and MAPKs signaling pathways (p-ERK, p-JNK, and p-p38). These findings indicated the inhibitory effect of crocin on airway inflammation and hyperreactivity in asthmatic mice (Xiong et al., 2015). Crocin (25 mg kg21; p.o.) treatment, after 16 days, significantly decreased the lung/body weight index, inflammatory cell counts in BALF, and lung TP content. Treatment also decreased pulmonary edema induced by

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OVA-allergic asthma-associated alterations. These results indicated that crocin protects against allergic asthma progression, which was associated with downregulation of inflammatory cytokine expression and restoration of oxidant/antioxidant homeostasis (Yosri et al., 2017). The antiinflammatory properties of crocin also have an effect on neuroinflammation. Xu et al. (2009) indicated that α-crocin blocked prostaglandin E2 (PGE2) production in LPS-stimulated RAW 264.7 cell lines. Crocin exhibits dual inhibitory activity against COX-1 and COX-2 enzymes. Crocin was also shown to inhibit the productions of PGE2 in LPS-challenged cells (Xu et al., 2009). Kim et al. (2014a,b) reported that crocin (500 μM) decreased LPS (0.1 μg mL21)-stimulated expression of iNOS by inducing heme oxygenase-1 (HO-1) expression via Ca21/calmodulin-dependent protein kinase 4-PI3K/Akt-Nrf2 signaling cascades in RAW 264.7 cells (Kim et al., 2014a). A neuroprotective effect of crocin (20 μM) treatment was also indicated by a decrease in the production of inflammatory mediators in cultured rat brain microglia as well as by the inhibition of LPS-induced apoptosis in organotypic hippocampal slice cultures (Nam et al., 2010). Crocin inhibited syncytin-1 and NO-induced astrocyte and oligodendrocyte cytotoxicity and reduced neuropathology in experimental autoimmune encephalomyelitis (EAE) with significantly fewer neurological impairments (Khazdair et al., 2015). Syncytin-1 has been contributed to oligodendrocyte death and neuroinflammation (Antony et al., 2004). Through EAE, the transcript levels of the endoplasmic reticulum (ER) stress genes XBP-1/s were increased and ER stress was shown to be closely related to inflammatory pathways (Marciniak et al., 2004). Administration of crocin (200 μM) on day 7 post-EAE induction suppressed ER stress and inflammatory gene expression in spinal cords and also reduced expression of ER stress genes XBP-1/s (Deslauriers et al., 2011). Neuroinflammation is related to the activation of microglia and release of cytokines. Microglial cells play a major role in immune and inflammatory responses in the central nervous system (CNS). Pretreatment with α-crocin (20 mg kg21) had protective effects on traumatic brain injury in mice, causing amelioration of neurological severity score and brain edema, reduction of microglial activation, and reduced release of several proinflammatory cytokines (Wang et al., 2015). Crocin (150 mg kg21) also improved locomotor function and mechanical behavior by reducing of calcitonin gene-related peptide, an important mediator of inflammation and pain, in rats with contused spinal cord injury (SCI; Karami et al., 2013). When observing crocin’s effect on gastrointestinal inflammation, it was found that crocin (50, 100, and 200 mg kg21, i.p.) significantly inhibited diazinon-induced elevation of AST, ALT, ALP, LDH, CPK, CPK-MB, GGT, and other inflammatory biomarkers such as TNF-α, 8-iso-prostaglandin F2a, and soluble protein-100 β in rat serum (Hariri et al., 2010). This agent also indicated inhibitory effect on the nuclear translocation of the NF-κB, p50, and p65 subunits. In addition, the protective effect of α-crocin against gastric lesion was mediated by its antiinflammatory properties, especially its inhibitory effect on COX (Xu et al., 2009). The potential inhibitory effects of α-crocin on mice colitis and colitis-related colon carcinogenesis in male rats induced by azoxymethane and dextran sodium sulfate showed the amelioration effect of crocin (50, 100, and 200 ppm). Crocin improves severe colorectal inflammation with mucosal ulcers, the inflammation score, and the presence of dysplasia. Including crocin as a dietary supplement also decreased the incidence of adenomas and multiplicities of colonic adenoma. In addition, feeding with crocin (100 and 200 ppm) decreased the severity of colitis in the mice; regenerative crypt cells covered and healed the mucosal ulcers. Crocin (200 ppm) also significantly decreased the mRNA expression of certain proinflammatory cytokines and inducible inflammatory enzymes including tumor necrosis factor α (TNF-α), interleukin-(IL-) 1β, COX-2, and inducible nitric oxide synthase in the colorectal mucosa (Kawabata et al., 2012). It has been reported that crocin (50 mg kg21 day21, i.p.) produced gastro-protective results against ethanol-induced gastric damages in rats. Crocin restored ethanol-induced lower levels of gastric juice mucin, mucosal PGE2 and IL-6. Crocin also significantly decreased TNF-α, MPO, and heat shock protein 70 mRNA, and protein levels and restored ethanol-altered mucosal levels of glutathione, malondialdehyde, and SOD activity. In addition, crocin significantly decreased mucosal apoptosis as revealed by significant downregulation of cytochrome c and caspase-3 mRNA expression, caspase-3 activity, and mitigated DNA fragmentation. The histological study also indicated that α-crocins, like omeprazole, ameliorated gastric lesions (El-Maraghy et al., 2015). In confirming crocin’s effect on other types of inflammation, the effects of α-crocin and diclofenac on local inflammation and pain induced by intraplantar injection of carrageenan in rats were compared. Crocins at the doses of 25, 50, and 100 mg kg21 and diclofenac at the dose of 10 mg kg21 ameliorated edema, decreased inflammatory pain responses (edema, cold allodynia, mechanical allodynia, and hyperalgesia), and decreased the number of neutrophils. The antiinflammatory and analgesic effects of crocin at 50 and 100 mg kg21 were similar to those of diclofenac 10 mg kg21 (Tamaddonfard et al., 2013). Tamaddonfard et al. (2012) also indicated antiinflammatory activity of α-crocin (100 mg kg21, i.p.) observed by a decrease in paw edema and infiltration of neutrophils in paw tissues induced by

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histamine (Tamaddonfard et al., 2012). In addition, α-crocin ameliorated paw swelling and inflammatory responses in arthritic rats. Crocin also demonstrated antiarthritic effects through the reduction of serum levels of enzymatic inflammatory mediators such as MMPs (MMP-13, MMP-3, and MMP-9), HAases and nonenzymatic inflammatory mediators (TNF-α, IL-1β, NF-κB, IL-6, COX-2, and PGE2) (Hemshekhar et al., 2012). In addition, crocin (20 mg kg21, p.o.) caused more marked effects on reduction of paw swelling and serum levels of COX-2 and PGE2 than ibuprofen, a nonsteroidal antiinflammatory drug (Hemshekhar et al., 2012).

25.2.3.2 Crocetin Crocetin is the major metabolite of crocin which displays different antiinflammatory activity, including effects on airway-inflammation. Pretreatment with crocetin (50 and 100 mg kg21, intragastric injection) reduced the LPS-induced acute lung injury (lung edema and histological disorders) in mice. Crocetin significantly decreased the inflammatory cell infiltration (especially that of neutrophils) and improved alveolar wall thickening, intraalveolar exudation, and interstitial edema. Furthermore, treatment with crocetin significantly increased SOD activity while decreasing lung MPO activity and mRNA levels of proinflammatory cytokines such as TNF-α, MCP-1, and IL-6 (which are markers of LPS-induced lung injury). In addition, crocetin at both doses reduced phospho-IκB expression and NF-κB activity in LPS-induced lung tissue damage. These results indicate that crocetin can protect LPS-induced acute lung injury in mice (Yang et al., 2012). When observing crocetin’s effect on neuroinflammation, it was found that crocetin treatment (20 μM) significantly suppressed LPS-induced nitrite release from microglial cells and reduced NF-κB activity in microglia. Crocetin also decreased LPS-stimulated production of proinflammatory cytokines (TNF-α and IL-1β) in cultured rat microglia cells and inhibited LPS-induced apoptosis in organotypic hippocampal slice cultures. This study suggests the neuroprotective effects of crocetin, which may be related to reduction of inflammatory cytokines production by activated microglia (Nam et al., 2010). The crocetin derivatives from Gardenia jasminoides inhibited NO production and showed the most potent antiinflammatory activity. Crocetin (20, 40, and 80 μM) also suppressed the expressions of protein and mRNA of iNOS and COX-2 in a dose-dependent manner in mouse macrophage cell line (RAW 264.7 cells) by western blot and RT
PCR analysis (Hong and Yang, 2013). Crocetin also has an effect on gastrointestinal inflammation. Oxidative stress and inflammation play an essential role in burn-induced intestinal injury and inhibition of inflammatory signaling attenuates the injured intestinal tract. Crocetin (100 and 200 mg kg21) when treating burn-induced small intestinal injuries in rats showed inhibited polymorphonuclear neutrophil accumulation in the small intestine and decreased the levels of TNF-α and IL-6. Burn injuries indicated by increasing levels of NF-κB and p65 showed reduction of burn-induced NF-κB activation when treated with crocetin. Mucosal ulceration and focal necrosis in the burn group were ameliorated by treatment with crocetin. These results suggested that crocetin treatment may protect against burn-induced small intestinal injury, possibly by inhibiting an inflammatory response (Zhou et al., 2015). Crocetin (50 mg kg21, intragastric injection) significantly resolved diarrhea and the disruption of colonic architecture. In crocetin-treated mice, the degree of neutrophil infiltration and lipid peroxidation in the inflamed colon was significantly reduced. Crocetin also reduced the levels of NO associated with the favorable expression of Th1 and Th2 cytokines and inducible NO synthase along with the downregulation of NF-kB. Crocetin (50 mg kg21) significantly decreased the severity of colitis by treating the necrosis of epithelium, distortion of crypts, destruction of glands, and infiltration of inflammatory cells in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. These results indicated that treatment with crocetin can result in decreased inflammation and edema through the inhibition of inflammatory cells (Kazi and Qian, 2009). When measuring the effect of crocetin on other types of inflammation, it was found that crocetin inhibited cellular inflammatory infiltration in tissue through the maintenance of vascular endothelial-cadherin expression, which is an adherence protein that has a key role in the control of vascular permeability in human umbilical vein endothelial cells and fibroblasts (Umigai et al., 2012). The effects of crocetin (1.0 μM) on the migration of vascular smooth muscle cells induced by advanced glycosylation end products decreased the levels of TNF-α, IL-6, MMP-2, and MMP-9 compared to the untreated group. Pretreatment with crocetin (50 mg kg21) reduced cardiac injury by reducing the levels of infarct size as well as the levels of TNF-α and IL-10 on myocardial damage after ischemia reperfusion (MIRI) when compared with untreated rats. These results showed that inhibition of inflammation is an important mechanism involved in the protective effect of crocetin against MIRI in rats (Wang et al., 2014). Inflammatory factors play an important role in cellular damage after shock and resuscitation. Crocetin (2 mg kg21, p.o.) improves postshock recovery of cellular adenosine triphosphate and increases overall survival in an experimental model of hemorrhagic shock. In a hemorrhagic shock model, crocetin displayed antiinflammatory effects by blocking TNF-α, IL-1β, and iNOS mRNA expression in the liver (Yang et al., 2006).

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25.2.3.3 Safranal Safranal is a derivative of saffron and has effects on airway inflammation. To confirm these prophylactic effects, lung inflammation in OVA-sensitized guinea pigs was studied. Safranal (4, 8, and 16 μg mL21) reduced inflammatory blood cells and ameliorated eosinophils and lymphocyte abnormalities after animal sensitization. In addition, the effect of safranal on eosinophil and lymphocyte count was significantly higher than hydroethanolic extract saffron and dexamethasone treatment (Bayrami and Boskabady, 2012). Treatment of sensitized animals with safranal (4, 8, and 16 μg mL21) significantly reduced serum levels of ET-1, histamine and TP compared to untreated sensitized animals and the effects of safranal were significantly higher than those of dexamethasone (Gholamnezhad et al., 2013). Treatment of sensitized animals with safranal (4, 8, and 16 μg mL21) in drinking water significantly improved pathological lung disorders, total amount of WBC, and serum histamine levels when compared to the sensitized group (Boskabady et al., 2012). The preventive effect of safranal in sensitized guinea pigs showed that tracheal responsiveness and levels of combined NO and nitrite in safranal-treated sensitized animals were significantly decreased when compared to the sensitized group (Boskabady et al., 2014). Safranal’s effect on neuroinflammation was also studied. The neuroprotective effects of safranal in SCI models showed that safranal (100 mg kg21, i.p.) decreased the immunoreactivity and expression of the inflammatory cytokines TNF-α, IL-1β, and p38 MAPK, as well as the expression of IL-10 after SCI. These results proposed antiinflammatory activity for safranal. Additionally, safranal reduced edema by decreasing the expression of aquaporin-4. These results suggest that safranal ameliorates neuronal function following SCI in rats, which were associated with antiinflammatory, antiapoptotic, and edema-attenuating effects (Zhang et al., 2015). Safranal (0.1 mg kg21, i.p.) ameliorated pain sensitivity and suppressed the expression of glial activation markers (GFAP and OX-42) and inflammatory cytokines (TNF-α and IL-1β) in ipsilateral dorsal horn of lumbar enlargement postsurgery. These results indicated that the antiallodynia effect of safranal after nerve injury is due to its inhibitory effect on glial activation and inflammatory cytokine generation in the CNS (Zhu and Yang, 2014). Safranal also has an effect on other types of inflammation. Safranal (0.025, 0.05, and 0.1 mL kg21, i.p.) and vitamin E prevented diazinon-induced increased levels of AST, ALT, ALP, LDH, CPK, CPK-MB, and GGT and other inflammatory biomarkers such as TNF-α, 8-iso-prostaglandin F2a and soluble protein-100 β in rats. These results indicated that sub-acute exposure to diazinon induces biochemical, enzymatic changes as well as inflammatory and neurotoxicity effects inhibited by safranal (Hariri et al., 2010). Safranal showed antiinflammatory and antinociceptive effects in writhing, formalin, and hot-plate tests in mice. Safranal (0.1, 0.3, and 0.5 mL kg21, i.p.) prevented the abdominal constrictions induced by acetic acid and, at 0.5 mL kg21, i.p., increased the pain threshold of mice against the thermal source 30 min after treatment. In the formalin test, safranal significantly reduced pain-related behaviors in phase I and phase II. Naloxone (2 mg kg21) did not prevent the antinociceptive effects of safranal (Hosseinzadeh and Shariaty, 2007). Safranal inhibited local inflammation and pain induced by intraplantar injection of carrageenan in rats. Safranal (0.5, 1, and 2 mg kg21) and diclofenac (10 mg kg21) ameliorated edema and decreased inflammatory pain responses (edema, cold allodynia, mechanical allodynia, and hyperalgesia) and the number of neutrophils. This effect of safranal (1 and 2 mg kg21) was similar to that of diclofenac (Tamaddonfard et al., 2013). The effects of safranal on inflammation in rats with experimental type 2 diabetes and obesity showed improvement in the inflammation of the plasma and pancreas tissue inhibited by a decrease in the levels of inflammatory cytokines (TNF-α, IL-18, and IL-1β). These results suggested that safranal may prevent diabetes and its complications via inflammation suppression (Hazman and Bozkurt, 2015). The antiinflammatory effects of saffron derivatives are shown in Table 25.3.

25.3

Immunomodulatory effects of saffron and its derivatives

The immune system protects the body against invading pathogens and the subsequently released mediators, both of which cause diseases. The imbalanced immune system had long been considered as the etiology of many disorders. The immune system may be disturbed by various physical, chemical, and biological agents, such as exposure to corrosive chemicals, excessive amounts of x-rays, sunlight and radioactive materials, extremes of cold and heat, or by pathogenic microorganisms and mechanical trauma (Gholamnezhad et al., 2015). In addition, there is a close relationship between oxidative stress, inflammatory response, and immune system. Excessive oxidative stress and inflammation may result in the activation and increase of CD81 lymphocyte, macrophage, neutrophils, and epithelial cells, as well as inflammatory mediators (IL-8, IL-1b, IL-6, TNFα, TGF-β, LTB4, and EGFR) (Balkissoon et al., 2011). Moreover, inflammatory pathways may be induced by dysregulation of the normal immune response (Domej et al., 2014). Finding a natural therapeutic agent is the aim of many studies for preventing and treating inflammatory and immune-mediated disease (Gholamnezhad et al., 2015).

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TABLE 25.3 Antiinflammatory effects different derivatives of saffron stigma. Constituent

Effect

Experimental model

References

Crocin

Suppressed airway inflammation and hyperreactivity, inhibited the expression of lung eotaxin and MAPKs

OVA-sensitized mice

Xiong et al. (2015)

Decreased the lung/body weight index, inflammatory cell counts in BALF, total lung protein content, and decreased pulmonary edema

OVA induced asthma in mice

Yosri et al. (2017)

Inhibited COX-1 and COX-2 enzymes and productions of PGE2

264.7 cell

Xu et al. (2009)

Decreased expression of iNOS via Ca /calmodulin-dependent protein kinase 4-PI3K/Akt-Nrf2

RAW 264.7 cells

Kim et al. (2014a)

Amelioration of neurological severity score and brain edema, decreased microglial activation, reduced release of several proinflammatory cytokines

Traumatic brain injury in mice

Wang et al. (2015)

Decreased AST, ALT, ALP, LDH, CPK, CPK-MB, and GGT, TNF-α, 8iso-prostaglandin in rat serum

Diazinon-induced rat

Hariri et al. (2010)

Amelioration of severe colorectal inflammation

AOM induced colitis in mice

Kawabata et al. (2012)

Downregulation of cytochrome c and caspase-3 mRNA expression, caspase-3 activity, mitigated DNA fragmentation

Ethanol-induced gastric damages in rats

El-Maraghy et al. (2015)

Ameliorated edema, inflammatory pain responses (edema, cold allodynia, mechanical allodynia, and hyperalgesia) and the number of neutrophils

Carrageenan induced local inflammation and pain

Tamaddonfard et al. (2013)

Reduction of enzymatic inflammatory mediators such as (MMP-13, MMP-3, and MMP-9) and nonenzymatic inflammatory mediators (TNF-α, IL-1β, NF-κB, IL-6, COX-2, and PGE2)

Rat model of arthritis

Hemshekhar et al. (2012)

Reduced the lung edema, histological disorders, reduced phosphoiκb expression, and NF-κB activity

LPS-induced lung injury

Yang et al. (2012)

Reduced nitrite release, NF-κB activity and production of proinflammatory cytokines (TNF-α and IL-1β)

Microglial cells

Nam et al. (2010)

Suppressed the expressions of protein and mRNA of iNOS and COX2

RAW 264.7 cells

Hong and Yang (2013)

Inhibited polymorphonuclear neutrophil accumulation in small intestine, decreased the levels of TNF-α and IL-6

Intestinal injuries in rats

Zhou et al. (2015)

Decreased severity of colitis (necrosis of epithelium, distortion of crypts, destruction of glands, and infiltration of inflammatory cells)

TNBS-induced colitis in rat

Kazi and Qian (2009)

Blocking TNF-α, IL-1β, and iNOS mRNA expression

Hemorrhagic shock model

Yang et al. (2006)

Reduced inflammatory blood cells and ameliorated eosinophils and lymphocyte abnormalities

OVA-sensitized guinea pigs

Bayrami and Boskabady (2012)

Decreased tracheal responsiveness, total NO, and nitrite

OVA-sensitized guinea pigs

Boskabady et al. (2014)

Decreased the expression of the inflammatory cytokines TNF-α, IL1β, and p38 MAPK, elevated the expression of IL-10

Spinal cord injury models

Zhang et al. (2015)

Ameliorated pain sensitivity, suppressed the expression of glial activation markers (GFAP and OX-42) and inflammatory cytokines (TNF-α and IL-1β)

Neuropathic pain

Zhu and Yang (2014)

21

Crocetin

Safranal

(Continued )

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TABLE 25.3 (Continued) Constituent

Effect

Experimental model

References

Increased the pain threshold of mice, reduced pain-related behaviors in phase I and phase II

Formalin and hotplate tests in mice

Hosseinzadeh and Shariaty (2007)

Ameliorated edema and decreased inflammatory pain responses

Carrageenan-induced inflammation

Tamaddonfard et al. (2013)

Amelioration of the inflammation in the plasma and pancreas tissue and decrease the levels of (TNF-α, IL-18, and IL-1β)

Diabetic nephropathy

Hazman and Bozkurt (2015)

Akt1, Activation a kinase; ALP, alkaline phosphatase; ALT, alanine transaminase; AOM, azoxymethane; AST, aspartate aminotransferase; BALF, broncho alveolar lavage fluid; COX, cyclooxygenase; CPK, creatine phosphokinase; GFAP, glial fibrillary acidic protein; GGT, gamma-glutamyl transferase; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinases; MMPs, matrix metalloproteinase; NF-κB, nuclear factor-kappa B; PGE2, prostaglandin E2; PI3K, phosphoinositide 3-kinase.

The effectiveness of some medical plants on immune system function has been demonstrated (Das et al., 2004). Medicinal plants, their derivatives, and plant-derived materials are believed to regulate immune system function and enhance the body’s natural resistance to infectious diseases. They may modulate proinflammatory cytokine and immunoglobulin secretion, histamine release, lymphocyte activation, phagocytosis, cellular coreceptor expression, and oxidative stress markers (Das et al., 2004; El-Beshbishy et al., 2012; Hassan et al., 2015). The major components of medicinal herbs include carotenoids, monoterpenes and polyphenols, which may enhance the immune system functions. Several antioxidants and radical scavenging have been reported in the experimental studies of the antiinflammatory and immunomodulatory properties of saffron and its derivatives. These will be reviewed in the next section.

25.3.1 Immunomodulatory effects of saffron extracts The modulatory effects of saffron’s different preparations and extracts on the immune system components (both humoral and cell mediated) have been shown in animal models as well as human studies. In the mouse model of EAE, the immunomodulatory effect of saffron ethanol extract has been shown. Saffron treatment significantly reduced the clinical symptoms of C57BL/6 mice with EAE as well as leukocyte infiltration in the spinal cord. Scientists believe that these results are related to the antioxidative properties of the plant and suggest that saffron may be potentially effective in multiple sclerosis treatment (Ghazavi et al., 2009). The effect of hydroethanolic extract of saffron has been evaluated on the serum ET-1, TP, and histamine levels of experimentally induced asthma in guinea pigs (OVA-sensitized animal). Results showed a significant decrease in serum concentration of ET-1, TP, and histamine in saffron treated animals (Boskabady et al., 2012; Gholamnezhad et al., 2013). Activating immune cells that generate free radicals is one of the main mechanisms by which medicinal plants such as saffron may affect the immune responses (Das et al., 2004). In Kanamarlapudi and Mohammad’s (2011) study, the effect of oral administration of a suspension of saffron stigmas at dose 50 and 100 mg kg21 had been evaluated on the immune system of mice. For the evaluation of humoral-mediated immunity, the following tests were completed: serum immunoglobulins, mice lethality test, and indirect hemagglutination test. For the evaluation of cell-mediated immunity, the following tests were completed: carbon clearance test, cyclophosphamide-induced neutropenia, neutrophil, and adhesion test. Saffron augmented the humoral-mediated immunity activity by increasing the circulating antibody titre, serum level of immunoglobulins and neutrophil adhesion to nylon fibers. Saffron also decreased mice mortality induced by lethal Pasteurellamultocida toxin. The minimum dose of saffron significantly increased the phagocytic index in carbon clearance test. However, saffron was not effective in preventing cyclophosphamide-induced neutropenia. These results indicated that saffron at low doses might have a stimulatory effect on humoral and cell-mediated immunity (Kanamarlapudi and Mohammad, 2011). There are controversial results about the effect of saffron on the Th1/Th2 limb of an immune response. Immunomodulatory impacts of saffron alcohol extract had been studied in sensitized mice with an injection of fresh sheep red blood cells (SRBC). The animals had been treated with saffron alcohol extract dose (1.56
50 mg kg21 p.o.). The results showed an elevation in agglutinating antibody titer as well as CD191 B cells number and IL-4 cytokine

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level, which indicated the stimulatory effect on Th2 subtype of humoral immunity. While the serum concentration of IL-2 and IFN-γ (Th1 cytokines) had been not changed significantly (Bani et al., 2011), it has been shown that saffron petals at a dose of 75 mg kg21 increased serum levels of IgG in SRBC-immunized rats. These results may confirm the effect of the plant on humoral antibody response in immunized animals. IFN-γ is the main cytokine to induce the B lymphocytes for IgG production, and it can be concluded that saffron may stimulate Th1 cell cytokine (IFN-γ) secretion (Babaei et al., 2014). In addition, the stimulatory effect of saffron on Th1 and the suppressive effect of saffron on Th2 cells in sensitized animals had been demonstrated. Treatment of ovalbumin-sensitized guinea pigs with saffron extract and dexamethasone decreased serum IL-4 concentration while increasing the IFN-γ levels and IFN-γ/IL-4 ratio. Therefore treatment of saffron extract in asthmatic guinea pigs may be as effective as dexamethasone in balancing Th1 and Th2 in the immune system (Byrami et al., 2013). Previously mentioned findings have been confirmed by the result of human studies. The effect of saffron macerated extract has been evaluated on cell viability and cytokine release of phytohemagglutinin (PHA) stimulated and nonstimulated peripheral blood mononuclear cells (PBMCs). Saffron showed an inhibitory effect on cell viability of both PHAstimulated and nonstimulated PBMC. It also increased IFN-γ and decreased IL-4 concentration in PBMC supernatants. These results indicated a stimulatory effect of the plant on the Th1/Th2 ratio (Boskabady et al., 2011). Moreover, prescribing saffron tablets (100 mg day21 for 6 weeks) to healthy men decreased the IgM level while increasing the IgG serum concentration (Kianbakht and Ghazavi, 2011). Therefore according to the overall findings of mentioned studies, saffron may possess therapeutic effects in inflammatory diseases with increased Th2 cytokine release, such as asthma.

25.3.2 Immunomodulatory effects of saffron derivatives 25.3.2.1 Immunomodulatory effects of crocin Crocin is one of main carotenoids and pharmacologically active components of saffron. Crocin is also responsible for the color of saffron. Cronic posseses different therapeutic properties, including effects on nervous system disorders (antidementia agent, antidepressant, anxiolytic, and aphrodisiac) as well as antioxidant, cardioprotective, and antitumor properties (Alavizadeh and Hosseinzadeh, 2014; Ben Salem et al., 2016). To confirm the neuroprotective effect of crocin, it was examined whether it could suppress the in vitro microglial activation as one of the brain immune and inflammatory responses. Results indicated that crocin inhibited LPS-induced inflammatory cytokines (TNF-α and IL-1β), NO and intracellular reactive oxygen species (ROS) overproduction, and NF-κB activation in microglial cell. It also decreased NO production in amyloid-β and IFN-γ stimulated microglia. These results suggest that crocin may have neuroprotective effects via decreasing the release of proinflammatory and neurotoxic factors from activated microglial cells (Nam et al., 2010). Crocin also may suppress the effect of TNF-α on neuronally differentiated PC-12 cells and blocked the TNF-α-induced expression of Bcl-XS and LICE mRNAs. In addition, it could improve the cytokine-induced reduction of Bcl-XL mRNA expression (Soeda et al., 2001). Crocin also caused suppressive effect on viper venom-induced proinflammatory cytokine elevation, and platelet and neutrophil apoptosis (Santhosh et al., 2016, 2013). The antiasthmatic effect of crocin in sensitized mice has been shown. Oral administration of crocin (100 mg kg21) significantly decreased airway hyperreactivity and inflammation as well as levels of BALF interleukins (IL-4, IL-5, IL13), lung eosinophil peroxidase, tryptase and serum OVA-specific IgE. Oral administration of crocin also suppressed the expression of lung eotaxin, p-JNK, p-ERK, and p-p38 in the OVA-sensitized mice (Xiong et al., 2015).

25.3.2.2 Immunomodulatory effects of crocetin Crocetin, similar to crocin, could suppress LPS-stimulated NF-κB activation and inflammatory cytokines, NO, and ROS overproduction in the microglial cells. It also decreased NO production in IFN-γ and amyloid-β activated microglial cells. These results confirmed a neuroprotective activity for crocetin and crocin, which may be exerted by their suppressing effects on production of proinflammatory and neurotoxic factors from activated microglial cells (Nam et al., 2010). It was showed that crocetin has a protective effect against LPS-induced acute lung injury in mice. Crocetin treatment (50 and 100 mg kg21) significantly decreased LPS-induced elevation of TNF-α and IL-6 protein and mRNA expressions, as well as MCP-1 in the lung tissue. Also, crocetin decreased phospho-IκB expression and NF-κB activity in lung tissue, which had been induced by LPS. These results showed that crocetin might modulate inflammatory and immune response pathways (Yang et al., 2012). The therapeutic effect of crocetin on an experimental model of

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TABLE 25.4 Immunomodulatory effects of saffron and its derivatives. Extract/ constituent

Effect

Experimental model

References

Stigma HEE

Increased Th1/Th2 balance

PHA-stimulated human lymphocytes

Boskabady et al. (2011)

Increased the Th2 response of humoral immunity resulting in the increase in agglutinating antibody titer

Mice

Bani et al. (2011)

Modulated blood inflammatory mediators (histamine, IL-4, IFN-γ, IFN-γ/IL-4 ratio, ET-1, and in blood)

OVA-sensitized guinea pigs

Gholamnezhad et al. (2013), Boskabady et al. (2012), Byrami et al. (2013)

Improved autoimmune encephalomyelitis via inhibiting leukocyte infiltration to CNS

Mice

Ghazavi et al. (2009)

Tablet

Decreased the IgM and increased the IgG serum concentration

Human

Kianbakht and Ghazavi (2011)

Petal EE

Increased humeral antibody response (IgG)

SRBC-immunized rats

Babaei et al. (2014)

Safranal

Modulated Th1/Th2 balance

PHA-stimulated/ nonstimulated PBMC

Feyzi et al. (2016)

Modulated blood cytokines, ET-1and TP

OVA-sensitized guinea pigs

Boskabady et al. (2012), Gholamnezhad et al. (2013)

Improved lung pathological disorders

OVA-sensitized guinea pigs

Boskabady et al. (2012)

Improved neurotoxicity induced by diazinon (decrease TNF-α, 8-iso-PGF2a, and SP-100 β)

Rats exposed to DZN

Hariri et al. (2010)

Reduced TNF-α level in diazinon treated groups, blocking TNF-α-induced Bcl-XS, and caspase-3 mRNAs, expression

Rat PC-12 cells

Soeda et al. (2001)

Inhibited viper venom-induced proinflammatory cytokines, platelet, and neutrophil apoptosis

Mice

Santhosh et al. (2013, 2016)

Decreased BALF IL-4, IL-5, IL-13; lung eosinophil peroxidase, tryptase, and serum OVA-specific IgE, and suppressed the expression of lung eotaxin, p-JNK, pERK, and p38

Sensitized mice

Xiong et al. (2015)

Protected acute lung injury by LPS (modulation IL-6, MCP-1, and TNF-α)

Mice

Yang et al. (2012)

Neuroprotective (decrease TNF-α, IL-1β from activated microglia)

Microglial cells exposed to LPS, IFN-γ, and amyloid-β

Nam et al. (2010)

Improved thrombosis (decreased platelet, plasma fibrinogen, protein C, glomerular fibrin deposition)

Rabbit exposed to bacterial endotoxin

Tsantarliotou et al. (2013)

Increased the levels of immunoregulatory proteins Foxp3 and protein 8-like 2 (TIPE2) in regulatory T (Treg) cells

Mice (OVA)-induced asthma

Ding et al. (2015)

Decreased the plasma level of maleic dialdehyde, polymorphonuclear cells, IL-1β, and TNF-α

Methylcholanthreneinduced uterine cervical cancer in mice

Chen et al. (2015)

Crocin

Crocetin

Bcl-XS, B-cell lymphoma-extra large; CNS, central nervous system; DZN, diazinon; EE, ethanolic extract; ET-1, endothelin-1; HEE, hydroethanolic extract; IgG, immonogluboline G; IFN-γ, interferon-γ; IL, interleukin; LPS, lipopolysaccharides; MCP-1, monocyte chemoattractant protein-1; OVA, ovalbumin; PC12, pheochromocytoma; PHA, phytohemagglutinin; SP-100B, soluble protein-100B; SRBC, sheep red blood cells; Th, T helper; TNF-α, tumor necrotic factor-α; TP, total protein; 8-iso-PGF2α, 8-iso-prostaglandin F2α.

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endotoxin-induced disseminated intravascular coagulation has been studied. Crocetin (3 mg kg21) treatment in rabbits restored changes in hemostatic indices such as platelet blood counts, plasma fibrinogen, protein C concentration, and fibrin deposition in the glomeruli (Tsantarliotou et al., 2013). The immunomodulatory effect of crocetin (100 μM) in mice (OVA)-induced asthma has been evaluated. Intranasal administration of crocetin increased the levels of two immunoregulatory proteins, Foxp3 and protein 8-like 2 (TIPE2), in regulatory T (Treg) cells (Ding et al., 2015). The effect of crocetin (10, 20, and 40 mg kg21, p.o.) on methylcholanthrene (MCA)-induced uterine cervical cancer in mice was also evaluated. Crocetin supplementation decreased the plasma level of maleic dialdehyde, polymorphonuclear cells, IL-1β, and TNF-α, which were elevated in MCA mice (Chen et al., 2015).

25.3.2.3 Immunomodulatory effects of safranal There are more than 34 ingredients in volatile compounds of saffron, some of which are terpenes and their esters. Among them, safranal is the major ingredient. In the diazinon immunotoxicity rat model, the protective effect of safranal has been indicated. Intraperitoneal injection of safranal (0.025, 0.05, and 0.1 mL kg21 three times per week), decreased the diazinon-induced TNFα elevation (Hariri et al., 2010). The balancing effect of safranal on serum cytokines (IFN-γ and IL-4) of sensitized guinea pigs has been shown. Safranal decreased the IL-4 concentration while increased the IFN-γ and IFN-γ/IL-4 ratio. Also, the effect of high concentration of saffron (4 μg kg21 day21) on reducing IL-4 and increasing IFN-γ and IFN-γ/IL-4 ratio was higher than the effect of dexamethasone (Boskabady et al., 2014). Moreover, safranal treatment reduced the serum histamine and ET-1 concentrations of experimental asthmatic guinea pigs (Boskabady et al., 2012; Gholamnezhad et al., 2013). The effect of safranal on immune system function and cellularity has been evaluated in the mice model of immunotoxicity (a delayed type of hypersensitivity (DTH) response and hemagglutination titer (HA)). The intrapreitoneal injection of three doses of safranal (0.1, 0.5, and 1 mL kg21) for 3 weeks did not significantly change the HA, DTH, proliferation response to PHA, blood/spleen cellularity IL-4, and INF-γ production or INF-γ/IL-4 ratios (Riahi-Zanjani et al., 2015). It has been indicated that safranal may affect cell viability and cytokine release of PHA-stimulated and nonstimulated PBMCs. All concentrations of safranal had an inhibitory effect on cell viability of PHA-stimulated PBMC, while only a high concentration of safranal showed inhibitory effect on nonstimulated cells. Two higher concentrations of safranal also increased IFN-γ and IFN-γ/IL-4 ratio in PBMC supernatants of both nonstimulated and stimulated cells. These results indicated a stimulatory effect of this constituent on the Th1/Th2 ratio (Feyzi et al., 2016). Different immunomodulatory effects of saffron and its derivatives are summarized in Table 25.4.

25.4

Conclusion

The antiinflammatory and immunomodulatory effects of saffron and its main derivatives—safranal, crocin, and crocetin—were demonstrated in inflammatory and immune dysregulation disorders in both experimental animal studies as well as in vitro experiments. These findings supported the preventive and therapeutic effects of saffron and its derivatives on these disorders. Saffron and its derivatives showed antiinflammatory and immunomodulatory effects through modulation of innate immunity (neutrophils, macrophage, and NK cells) as well as acquired immunity component (inflammatory and antiinflammatory cytokines, B cell, and Th1/Th2). Therefore it is suggested that saffron and its derivatives safranal, crocin, and crocetin may be of therapeutic value in diseases management. However, more clinical investigations are needed to confirm these antiinflammatory and immunomodulatory effects of the saffron plant and its derivatives in clinical practice. Furthermore, the safety assessments of long-term administration of saffron and its derivatives should be examined to determine probable toxic effects in human.

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Nam, K.N., Park, Y.M., Jung, H.J., Lee, J.Y., Min, B.D., Park, S.U., et al., 2010. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. Eur. J. Pharmacol. 648, 110
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358. Premkumar, K., Thirunavukkarasu, C., Abraham, S.K., Santhiya, S.T., Ramesh, A., 2006. Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agents in mice. Hum. Exp. Toxicol. 25, 79
84. Riahi-Zanjani, B., Balali-Mood, M., Mohammadi, E., Badie-Bostan, H., Memar, B., Karimi, G., 2015. Safranal as a safe compound to mice immune system. Avicenna J. Phytomed. 5, 441
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8330. Yang, R., Tan, X., Thomas, A.M., Shen, J., Qureshi, N., Morrison, D.C., et al., 2006. Crocetin inhibits mRNA expression for tumor necrosis factor-α, interleukin-1β, and inducible nitric oxide synthase in hemorrhagic shock. J. Parent Enteral Nut. 30, 297
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60.

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Zhang, R., Zhi-Yu, Q., Xiao-Yuan, H., Zhen, C., Jun-Ling, Y., Hamid, A., 2009. Comparison of the effects of crocetin and crocin on myocardial injury in rats. Chin. J. Nat. Med. 7, 223
227. Zhang, C., Ma, J., Fan, L., Zou, Y., Dang, X., Wang, K., et al., 2015. Neuroprotective effects of safranal in a rat model of traumatic injury to the spinal cord by anti-apoptotic, anti-inflammatory and edema-attenuating. Tissue Cell 47, 291
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107. Zhu, K.J., Yang, J.S., 2014. Anti-allodynia effect of safranal on neuropathic pain induced by spinal nerve transection in rat. Int. J. Clin. Exp. Med. 7, 4990
4996.

Further reading Ochiai, T., Shimeno, H., Mishima, K., Iwasaki, K., Fujiwara, M., Tanaka, H., et al., 2007. Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo. Biochim. Biophys. Acta 1770, 578
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Chapter 26

Effectiveness of saffron on memory function, learning ability, and epilepsy Hamid-Reza Sadeghnia1,2, Arezoo Rajabian3 and Seyed-Mahmoud Hosseini2,4 1

Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 2Division of Neurocognitive Sciences,

Psychiatry and Behavioral Sciences Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 3Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran, 4Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 26.1 Introduction 26.2 In vitro and preclinical studies 26.2.1 Memory and learning skills 26.2.2 Oxidative stress 26.2.3 Alzheimer’s disease

26.1

423 423 423 425 426

26.2.4 Seizure 26.3 Clinical studies 26.4 Conclusion References

427 427 427 428

Introduction

The stigma of Crocus sativus, commonly referred to as saffron, is cultivated in several countries including Iran, India, and Greece (Rios et al., 1996). It has been used in Persian and Indian traditional medicine for the central nervous system (CNS) disorders including dementia, cognitive dysfunction, and mental diseases (Akhondzadeh and Maleki, 2007; Hatami, 2004; Purushothuman, 2015) and also as an anticonvulsant remedy (Zargari, 1995). Phytochemical analysis has revealed that saffron stigma mainly contains the carotenoid pigments crocin (crocetin di-gentiobiose ester) and crocetin; the odor responsible terpenes, especially safranal; and the bitter glucoside picrocrocin, which is responsible for saffron’s flavor (Gohari et al., 2013). Potential neuroprotective and anticonvulsant properties of saffron as well as its effectiveness on learning and memory processes have been proposed in several experimental and clinical evidence as discussed here.

26.2

In vitro and preclinical studies

26.2.1 Memory and learning skills Learning refers to the process of acquiring knowledge, whereas memory, one of the most crucial mental capabilities, is retention and retrieval of acquired knowledge (Kupfermann, 1993). Hippocampal long-term potentiation (LTP) is a form of activity-dependent synaptic plasticity that is considered as a mechanism underlying learning and memory via storing information in the CNS (Akhondzadeh, 1999). Ethanolic extract of saffron (125 and 250 mg kg21 and crocin 50
200 mg kg21, p.o.), but not crocetin, were found to counteract ethanol (30%, 2, and 10 mL kg21, p.o.)-induced performance deficits, impairments of learning and memory, and LTP suppression, in vitro and in vivo, in a dose-dependent manner (Sugiura et al., 1995a,b,c). As indicated in a study conducted by Zhang et al. (1994) the ethanolic extract of saffron with single doses of 125, 250, and 500 mg kg21 ameliorated the ethanol-induced impairments of learning and memory acquisition and retrieval in step-through and step-down tests in the mice orally administrated. Inhibition of ethanol-induced impairment of Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00027-7 © 2020 Elsevier Inc. All rights reserved.

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hippocampal synaptic plasticity was suggested to be related to the antagonistic effect of crocin on synaptic potentials mediated by N-methyl-D-aspartate (NMDA) receptors in the dentate gyrus of rat hippocampal slices (Abe et al., 1998; Akhondzadeh, 1999; Kumar et al., 2011; Sugiura et al., 1994, 1995a,b). Crocetin gentiobiose glucose ester and crocetin di-glucose ester, which are analogs of crocin, showed lower ameliorating activity on LTP-blocking effect of ethanol than crocin. The activity was proportional to the number of glucoses. The beneficial effects on learning and memory could be attributed to crocin, the actual active component in saffron, possessing four glucoses in a molecule (Sugiura et al., 1994). Another study also confirmed the protective effect of saffron aqueous extract (0.0025
0.56 g kg21), crocin (50 and 200 mL kg21), and safranal (0.2 mL kg21), which were administrated for 5 days, against scopolamine-induced learning impairment in rats using Morris water maze task (Hosseinzadeh and Ziaei, 2006). Investigation of the effects of saffron extract (30 and 60 g kg21) and the active constituent, crocin (15
30 mg kg21), on recognition and spatial memory revealed an antagonistic effect against extinction of recognition memory and attenuated scopolamine-induced spatial memory performance deficits in the novel object recognition test (NORT) and the radial water maze task in rats. Crocin (30 mg kg21) was able to antagonize the scopolamine-induced reference and working memory deficits (Kumar et al., 2011; Pitsikas and Sakellaridis, 2006; Pitsikas et al., 2007). The results obtained from a study demonstrated the effectiveness of crocin (30 mg kg21, i.p. for 3 weeks) in antagonizing performance deficits induced by intracerebroventricular (i.c.v.) administration of streptozocin (STZ), in the passive avoidance and the spatial Y-maze memory procedures in rats (Khalili and Hamzeh, 2010). Also, Naghizadeh et al. (2013) using the same procedure, demonstrated that oral administration of crocin (100 mg kg21) effectively attenuated spatial memory deficit and oxidative stress induced by STZ (3 mg kg21, i.c.v.) in rats. The synergistic effects of the combination of Nardostachys jatamansi extract (200 mg kg21), crocetin (25 mg kg21), and selenium (0.05 mg kg21) in a model of experimental dementia was investigated (Khan et al., 2012). Intracerebroventricular infusion of STZ resulted in induction of oxidative stress and depletion of endogenous antioxidants along with cognitive loss due to failure of cellular energetics (Nitsch and Hoyer, 1991). In the combinationpretreated rats, the cognitive performance was restored, through attenuation of oxidative stress (Khan et al., 2012). In addition, another study also showed the preventive effect of aqueous saffron extract (60 mg kg21; i.p. for 3 weeks) and safranal (60 mg kg21; i.p. for 3 weeks) against STZ-induced learning, memory, and cognitive impairments in rats, using passive avoidance paradigm Y-maze task. Crocin reversed transfer latency paradigm in the elevated plus-maze, an index of learning and memory, in STZinduced diabetic rats (Tamaddonfard et al., 2013). Considering the presence of insulin and insulin receptors in the hippocampus, which are involved in learning and memory (Zhao et al., 2004), and the fact that hyperglycemia induces neuronal degeneration through oxidative stress following diabetes (Li and Sima, 2004), the authors suggested that crocin improved cognitive deficits via antihypoinsulinemic, antihyperglycemic, antioxidant, and neuroprotective properties (Tamaddonfard et al., 2013). As in the previous study, treatment of diabetic rats with crocin (15, 30, 60 mg kg21, i.p. for 6 weeks) significantly improved spatial memory impairment accompanied by cerebral oxidative damage in the Morris water maze paradigm (Ahmadi et al., 2017). A study was also performed to evaluate the effect of hydroalcoholic extract of saffron on D-galactose and sodium nitrite (NaNO2)-induced amnestic mice. Prolonged systemic administration of the extract (30 mg per kg day21, i.p. for 15 days) along with amnesia induction exerted preventive and ameliorative effects against learning and memory impairments, examined by one-way passive and active avoidance tests (Dashti et al., 2012). A single posttraining injection of crocin (15 and 30 mg kg21) prevented the recognition memory deficits produced by the NMDA glutamate receptor antagonist, ketamine-disrupted rats in the NORT (Georgiadou et al., 2014). Safranal was found to reduce the extracellular concentrations of excitatory amino acids, glutamate, and aspartate in the rat hippocampus following kainate treatment (Hosseinzadeh et al., 2008). Crocin (2 mg kg21, i.p.) inhibited ketamine-induced retrograde amnesia and restored the passive avoidance response in rats (Yousefvand et al., 2016). Reduced extracellular glutamate levels by saffron and its constituents might be involved in the beneficial effect of crocin on ketamine-induced behavioral deficits. In the same way, C. sativus hydroethanolic extract has been shown to prevent glutamatergic synaptic transmission through interacting with NMDA receptors, in a concentration-dependent manner (Berger et al., 2011). Investigation of the effect of aqueous saffron extract on morphine-induced memory impairment revealed that treatment of the mice by 150 and 450 mg kg21 of the extract increased the time latency for entering the dark compartment at 24 and 48 hours after the training and retention trials in the passive avoidance task (Naghibi et al., 2012). Haghighizad et al. (2008) also showed the beneficial effects of saffron at lower doses (10, 30, and 50 mg kg21, i.p.) on spatial learning and memory parameters in the animals receiving morphine using Morris water maze task.

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The role of several neurotransmitter systems in the beneficial effects of saffron and its constituents on learning and memory have been proposed, including glutamatergic, cholinergic, GABAergic, and dopaminergic systems. As mentioned earlier, NMDA receptors may be involved in the beneficial effect of saffron or its constituents on memory (Abe et al., 1998, 1999). Saffron could restore cognitive dysfunction caused by cholinergic blockade (Pitsikas and Sakellaridis, 2006; Pitsikas et al., 2007). Cognitive impairments may occur following epilepsy and alterations in normal synaptic transmission and LTP process (Sgobio et al., 2010). Protective effect of safranal, an active constituent of saffron, against pentylenetetrazoleinduced seizures has been proposed to be mediated via modulation of GABAergic system (Hosseinzadeh and Sadeghnia, 2007). Administration of crocin (5, 10, and 20 mg kg21, p.o.) improved learning and memory impairments in pentylenetetrazol-kindled mice. These findings were supported by reduced neuronal damage in the hippocampal pyramidal layer (Mazumder et al., 2016). A study was also performed to investigate whether crocin could counteract nonspatial and spatial recognition memory impairments induced by apomorphine, a dopamine receptor agonist, in rats (Pitsikas and Tarantilis, 2017). Administration of crocin (15 and 30 mg kg21) prevented apomorphine-induced performance deficits in the NORT but not spatial recognition memory deficits produced in the novel object location task. The same investigators reported that crocin could antagonize spatial memory deficits produced by a muscarinic receptor antagonist (scopolamine) in rats (Pitsikas et al., 2007). As mentioned earlier, crocin also improved recognition memory deficits caused by dysfunction of the glutamatergic system (Georgiadou et al., 2014). Based on these results, saffron and its constituents are capable of antagonizing the deleterious effects of ethanol, scopolamine, ketamine, morphine, and apomorphine on learning and memory acquisition.

26.2.2 Oxidative stress Oxidative damage to lipids, proteins, and nucleic acids contributes to cytotoxicity and dysfunction of neurotransmitters/ neurotrophin systems and is considered as a critical pathogenic factor in the several neurodegenerative disorders accompanied by progressive cognitive deficits (Forster et al., 1996; Muriach et al., 2010). It has been well documented that the memory-enhancing effects of saffron and its constituents may be related to its antioxidant potential. Some in vitro and in vivo preclinical experiments propose a protective role for saffron extracts, crocin and safranal, in cerebral ischemia. Saffron stigma aqueous extract (100 mg kg21, p.o.) modified the pathological alterations following ischemia induced by middle cerebral artery occlusion and reperfusion in rat. Protective effects of the extract against behavioral deficits and neural cell death were related to the restoring glutathione and antioxidant enzymes, through improving energy metabolism during ischemia, and partly through inhibition of lipid peroxidation (Saleem et al., 2006). Similarly, safranal (727.5, 363.75, 145.5 mg kg21, i.p.) dose-dependently ameliorated global and focal cerebral ischemia-reperfusion injury in rat hippocampus as indicated by modified oxidative stress indices (Hosseinzadeh and Sadeghnia, 2005; Sadeghnia et al., 2017). Furthermore, crocin (20 mg kg21, i.p.) counteracted reperfusion-induced oxidative/nitrative injury in a mice model of transient global cerebral ischemia (Zheng et al., 2007). Administration of saffron extract (1 mg kg21 day21, i.p.) to rats exposed to the mitochondrial toxin, 3nitropropionic acid, induced neuroprotective effects against impairment of energy metabolism and oxidative stress (DelAngel et al., 2006). Another study confirmed potential therapeutic application of safranal (72.75, 145.5, and 291 mg kg21, i.p.) using a model of neurodegenerative disorder induced by quinolinic acid in rat (Sadeghnia et al., 2013). In an in vitro study conducted by Mehri et al. (2012) crocin (10
050 μM) protected PC12 cells from acrylamideinduced apoptotic cell death, at least partly by inhibition of intracellular ROS production. The protective effects of crocin (12.5, 25, and 50 mg kg21, i.p.) were further confirmed by improved behavioral index and histopathological damages in the rat cerebral cortex and cerebellum following exposure to acrylamide (50 mg kg21, i.p.), in a dosedependent manner (Mehri et al., 2015). Saffron and crocin have been introduced as potent antioxidants (Abe and Saito, 2000; Asdaq and Inamdar, 2010). The results of several studies demonstrated that hydroalcoholic extract of saffron (100
250 mg kg21, i.p.) and crocin (5
30 mg kg21, i.p.) ameliorated spatial cognitive deficits following chronic cerebral hypoperfusion in rats (Ghaeni et al., 2012; Hosseinzadeh et al., 2012). Chronic cerebral hypoperfusion impaired the antioxidant defense system of the cortex and hippocampus inducing neuronal degeneration and death (Xu et al., 2010). Therefore, the memory-enhancing effect of saffron extract and crocin may due to antioxidant and free radical scavenging properties (Ghaeni et al., 2012; Hosseinzadeh et al., 2012).

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Age-related cognitive and memory deficits can be due to oxidative stress and impairment of cholinergic function. Administration of saffron resulted in significant improvement of learning and memory in both adult and aged mice, as evidenced by the passive avoidance paradigm, and the effect was accompanied by amelioration of oxidative stress and caspase-3 activity. Protective effects of saffron (1
250 μg mL21), crocetin, and safranal (1
125 μM) were also demonstrated against H2O2-induced toxicity in human neuroblastoma SH-SY5Y cells, in vitro (Papandreou et al., 2011). A study was performed to evaluate the protective effects of saffron extract and its active constituent, crocin, against oxidative stress and loss of spatial learning and memory induced by chronic stress in rats. Administration of saffron extracts (30 mg kg21, i.p.) or crocin (15
30 mg kg21, i.p) over a period of 21 days significantly blocked both oxidative stress and impairment of spatial learning and memory retention (Ghadrdoost et al., 2011). Considering that cognitive deficits affect a large proportion patients with multiple sclerosis (MS) (Rocca et al., 2015), a study was designed to evaluate the effects of saffron extract on learning and memory impairments in an animal model of MS. The observations demonstrated that the extract (5 and 10 μg rat21) alleviated detrimental effects of intrahippocampal injection of ethidium bromide on spatial memory and learning via modulation of oxidative stress markers (Ghaffari et al., 2015).

26.2.3 Alzheimer’s disease Alzheimer’s disease (AD) is clinically characterized by cognitive and memory deterioration. Formation of neuritic amyloid plaques and neurofibrillary tangles of tau protein in neurons are the main molecular process underlying AD. Oxidative stress may be involved in promotion of Aβ fibril formation and deposition (Ballard et al., 2011). There is some evidence that saffron extracts may inhibit beta-amyloid aggregation, a key step in the pathogenesis of AD, in animal experimental models (Papandreou et al., 2006). The measurement of the thioflavine T-based fluorescence of Aβ1-40 showed that the water:methanol (50:50, v/v) extract of C. sativus prevented Aβ fibrillogenesis in concentration and time-dependent manners, suggesting the possible use of saffron extract or its constituents in prevention of aggregation and deposition of Aβ in the human brain (Papandreou et al., 2006). The loss of cholinergic neurons due to Aβ and tau formation correlate with the cognitive deficits (Perry et al., 1978). In vitro enzymatic and molecular docking studies revealed the inhibitory action of saffron extract and its constituents, crocetin, dimethylcrocetin, and safranal on acetylcholinesterase, resulting in an increase in the synaptic acetylcholine levels (Geromichalos et al., 2012). The neuroprotective effects of transcrocetin against Aβ42-induced toxicity in hippocampal-derived cells was also demonstrated (Kong et al., 2014). Consistent with this finding, other studies also suggested that transcrocetin (at low micromolar concentrations) enhanced Aβ42 degradation in monocytes isolated from AD patients through the upregulation of the lysosomal protease cathepsin B (Tiribuzi et al., 2017). Another study also indicated the neuroprotective properties of crocetin against Aβ1-42-induced cytotoxicity in murine HT-22 hippocampal neuronal cells through counteracting oxidative stress (Yoshino et al., 2014). Modulation of microglial activity has been shown to be effective in neurodegenerative conditions. Crocin and crocetin suppressed LPS-induced production of nitric oxide, intracellular ROS, tumor necrosis factor-α, interleukin-1β and NF-κB activation as well as NO release from the cultured rat brain microglial cells stimulated with interferon-γ and amyloid-β (Nam et al., 2010). Batarseh conducted an in vitro and in vivo study to investigate the mechanisms by which saffron and crocin exert their protective effects against AD. This study showed that the ethanolic extract of saffron and crocin reduced Aβ load and related brain pathological changes by enhancing Aβ clearance pathways including blood-brain barrier (BBB) clearance, enzymatic degradation and ApoE clearance pathway, and also by decreasing neuroinflammation (Batarseh et al., 2017). Implication of neurotoxicity induced by aluminum, as an environmental factor, in the pathogenesis of neurodegenerative diseases has been reported (Domingo, 2006). It was suggested that the protective properties of aqueous saffron extract (200 mg kg21) and honey syrup against neurotoxicity following administration of aluminum chloride may be due to antioxidant activities (Shati et al., 2011). In a study conducted by Linardaki et al. (2013) the potential of saffron extract (60 mg kg21, i.p. 6 days) to counteract the changes accompanying aluminum neurotoxicity was investigated. Although short-term coadministration of saffron failed to reverse aluminum-induced impairment of learning/memory ability of mice in passive avoidance tests it considerably inhibited the aluminium-induced neurotoxicity and oxidative stress and restored the monoamine oxidase and acetylcholinesterase activities in the whole brain and cerebellum.

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26.2.4 Seizure Experimental studies performed in rodents using pentylenetetrazole (PTZ) and the maximal electroshock seizure (MES) tests demonstrated anticonvulsant activity of aqueous and ethanolic extracts of saffron (0.8 and 2 g kg21, i.p.) and safranal (0.15 and 0.35 mL kg21, i.p.), as indicated by the delayed the onset of tonic convulsions and the reduced seizure period and mortality. But crocin (200 mg kg21, i.p.) failed to show any protective effect (Hosseinzadeh and Talebzadeh, 2005; Khosravan, 2002). Intraperitoneal administration of safranal (72.75, 145.5, and 291 mg kg21) dose-dependently decreased the frequency of minimal clonic and generalized tonic-clonic seizures as well as mortality upon seizures induced by PTZ. Considering that flumazenil and naloxone could antagonize the anticonvulsant effect of safranal, it is suggested that the GABAA-benzodiazepine receptor complex may be involved in the protective effects of safranal against the tonic and clonic phases of PTZ-induced seizures (Hosseinzadeh and Sadeghnia, 2007). Consistent with the previous study, safranal exhibited dose-dependent antiabsence seizure activity, elicited by either γ-butyrolactone, baclofen, picrotoxin (1.5 mg kg21), bicuculline (5 mg kg21), or low dose of PTZ in C57BL/6 mice, suggesting modifications on the benzodiazepine binding sites of the GABAA receptor complex (Sadeghnia et al., 2008). Similarly, it was revealed that both ethanolic (250 and 500 mg kg21, i.p.) and aqueous (200, 400, and 800 mg kg21, i.p.) extracts of C. sativus reduced convulsions induced by PTZ or MES in rats (Dalu and Shanker, 2017; Sunanda et al., 2014).

26.3

Clinical studies

Several trials have been carried out to assess the effects of saffron in humans suffering from memory disorders. Fortysix patients with the mild-to-moderate AD randomly received saffron capsules (15 mg twice per day, p.o.) or placebo for a 16-week clinical trial study. Psychometric measures, including AD assessment scale-cognitive subscale (ADAScog), and clinical dementia rating scale-sums of boxes (CDR), were performed to monitor the global cognitive profile. In this double-blind, placebo-controlled study, saffron produced a significantly better outcome on cognitive function than placebo, while there were no significant differences in the two groups in terms of observed adverse events (Akhondzadeh et al., 2010a). A clinical trial study was carried out in 46 patients (adults 55 years of age or older) suffering from mild-to-moderate AD. In this multicenter, double-blind controlled trial, the patients randomly received saffron (30 mg day21, p.o.) or an acetylcholine esterase inhibitor, donepezil (10 mg day21, p.o.), for 22 weeks. The findings of this phase II study suggested that saffron was safe and well tolerated and its efficacy (as measured by ADAS-cog and CDR) in the treatment of mild-to-moderate AD is similar to donepezil (Akhondzadeh et al., 2010b; Pitsikas, 2015). A clinical trial was also performed aiming to compare the efficacy and safety of C. sativus with memantine, an NMDA receptor antagonist, in patients with moderate-to-severe AD. In this double-blind clinical trial, 68 patients randomly received saffron (30 mg day21, p.o.) or memantine (20 mg day21, p.o.) for 12 months. The patients were evaluated every month using the Severe Cognitive Impairment Rating Scale and Functional Assessment Staging, in addition to recording the probable adverse events. The efficacy and safety of C. sativus for reducing cognitive deterioration was comparable to memantin (Farokhnia et al., 2014). Another study was carried out in 30 patients with mild-to-moderate AD to assess the effect of combination of honey, saffron, and sedge (Cyperus rotundus) on cognitive dysfunction. The cognitive status was measured by the standard scale ADAS-cog. Based on the findings, the investigators concluded that the combination treatment had no more pronounced effect than placebo on cognitive dysfunction (Jivad et al., 2015). Assessment of the effect of saffron ethanolic extract on the visual short-term memory of 20 volunteers for 3 weeks, in a regular double-blind manner, revealed improvement in short-term memory capacity (Ghodrat et al., 2014).

26.4

Conclusion

This chapter summarized different in vitro and in vivo studies looking at the therapeutic potential of saffron and its active constituents (crocin, crocetin, and safranal) alone or in combination with other compounds for cognitive impairments and neurodegenerative and neurological disorders such as epilepsy. The results of the investigations showed that saffron and its active constituents induced pharmacological effects including antiinflammatory, neuroprotective, antioxidant, and cognition-enhancing activities. The beneficial properties of these phytochemicals are attributed, at least in part, to the interaction with GABA, cholinergic, glutamatergic, and

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dopaminergic systems. Therefore, saffron and its active constituents may be considered effective remedies for treatment of neurodegenerative and neurological disorders. To establish the therapeutic utility of these phytochemical, more human trials should be carried out.

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707. Sugiura, M., Saito, H., Abe, K., Shoyama, Y., 1995a. Ethanol extract of Crocus sativus L. antagonizes the inhibitory action of ethanol on hippocampal long-term potentiation in vivo. Phytother. Res. 9 (2), 100
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94.

Chapter 27

Antidepressant and antianxiety properties of saffron Seyed Ahmad Mohajeri1,2, Samaneh Sepahi3 and Adel Ghorani Azam4 1

Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran, 2Department of

Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, 3Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran, 4Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 27.1 Introduction 27.2 Nervous system 27.3 Depression and anxiety 27.3.1 Pathophysiology of depression and anxiety 27.3.2 Epidemiology of depression and anxiety 27.3.3 Causes of depression and anxiety 27.4 Antidepressants 27.4.1 Classification of antidepressants and antianxiety drugs 27.4.2 Pharmacokinetics 27.4.3 Mechanism of action of antidepressants and antianxiety drugs 27.4.4 Possible side effects of antidepressants 27.5 Traditional medicine for the treatment of depression and anxiety 27.6 Saffron

27.1

431 431 432 432 432 433 433 433 434 434 434 435 435

27.6.1 Chemical compounds of saffron 435 27.6.2 Pharmaceutical applications of saffron in traditional medicine 436 27.6.3 Pharmacology of saffron and its active ingredients 436 27.6.4 In vivo studies on the effects of saffron and its active compounds on depression and anxiety 436 27.6.5 Antidepressant and anxiolytic properties of saffron in clinical practice 437 27.6.6 Mechanistic pathway for antidepressant and antianxiety effects 438 27.6.7 Effect of saffron and saffron compounds on nervous system diseases 438 27.7 Conclusion 440 References 441

Introduction

Many drugs with wide range of efficiency are used to treat depression and anxiety, most of which have side effects. Medicinal plants due to owning many therapeutic potentials and also acceptance by the patients are considered as new treatment modality for management of various diseases. Saffron, typically used as spice and flavoring agent, has many pharmacological properties, in particular antiinflammatory, antioxidant, antidepressant, and antianxiety effects. In this chapter, the antidepressant and antianxiety properties of saffron and its major constituents are discussed based on recent evidence. The likely mechanistic pathways of saffron, safranal, and crocin will also be discussed.

27.2

Nervous system

The nervous system is involved in almost all bodily processes and is divided into the central (CNS) and peripheral nervous systems (PNS). The CNS consists of the brain and spinal cord, and the PNS consists of sensory organs and sensory nodes (Kim et al., 2016a; Robson et al., 2017). The brain is part of the CNS, which is surrounded by the skull. This part consists of the brain stem, cerebral cortex, cerebellum, corpus callosum, thalamus, hypothalamus, and hippocampus. The hippocampus is a sensitive part of the brain associated with the limbic system, which is considered as the Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00028-9 © 2020 Elsevier Inc. All rights reserved.

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center for short-term, long-term, and spatial memory, as well as learning. The hippocampus, which has a seahorse-like shape, is located in the inner fold of the lower midbrain under the cerebral cortex called temporal lobe. The hippocampus has two parts, one part on each side of the head. The hippocampus is a sensitive part of the brain that can be negatively affected by many different conditions. It has been shown that diseases such as epilepsy, Alzheimer’s, and depression can induce damage to the hippocampus and influence hippocampal function. Severe depression can also cause atrophy of the hippocampus leading to total hippocampal volume loss (Geerlings and Gerritsen, 2017; Liu et al., 2017; Pinar et al., 2017). Physical and mental health of the human are in close relationship, and any defect in mental state affects the physical health. While the symptoms of a physical illness are usually obvious by physical examination, the symptoms of mental illness are reflected in emotions, feelings, and behavior, which are not always obvious. Hence, the symptoms of mental disorders may not always be diagnosed properly (Kim et al., 2016b; Perini et al., 2017). There is considerable evidence indicating that stress negatively affects the hippocampus. For example, patients with Cushing’s disease exhibit symptoms associated with high levels of cortisol (known as the stress hormone) such as hippocampal volume loss. Losing the volume of the hippocampus has been reported among people in whom depression began before the age of 21, as well as in those who experience chronic depression. Long-term depression can damage the brain. Loss of hippocampus volume has not been observed in patients who have experienced only short bouts of depression (Ragnarsson et al., 2017; Zimmerman et al., 2016).

27.3

Depression and anxiety

27.3.1 Pathophysiology of depression and anxiety Depression is the third most common illness and the first chronic disease, which affects the life of individuals. Depression is often caused by constant stress during daily life (Gold and Chrousos, 2002). Depression, like other mental health disorders, imposes high costs on the healthcare system. It is the most common mental illness, and it is estimated that about 10% of people have experienced at least one period of depression in the past year. Symptoms of depression include dissatisfaction, loss of energy and interest in activities, low self-confidence, feelings of sadness or depression, mood and appetite changes, changes in sleep patterns, and decreased libido. Several drugs have been used to treat depression, including selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs), and monoamine oxidase inhibitors (MAOIs), which treat depression through induction of chemical changes in the brain (Ferguson, 2001; Hillhouse and Porter, 2015). Depression is a serious illness associated with symptoms at the physiological, psychological, and behavioral levels (Hosseinzadeh and Khosravan, 2002). It has been shown that the activity of gene-encoding neurokinin, known as the brain-derived neurotrophic growth factor, decreases following chronic stress (Koolhaas et al., 1997). In addition, stress may negatively affect the process of nerve regeneration (Sudweeks and Yakel, 2000). Therefore, chronic stress causes changes in the functioning of neurons, which can ultimately lead to cell death and subsequently depression (Koolhaas et al., 1997; Sudweeks and Yakel, 2000). MRI images of the brains of depressed people have shown reduced volume of specific structures of the limbic system including the amygdala, prefrontal cortex, and hippocampus. Reduced density and size of neurons and glial cells have also been seen in the brains of people with depression. Reduced volume of the hippocampus can be attributed to neural cell death, changes in neurons and glial cells, and decreased neurogenesis. Ultimately, these neuronal changes cause depression and cognitive impairment (Campbell and MacQeen, 2004; Fava et al., 1997). People who suffer from depression may experience weight gain or weight loss, sleepiness or insomnia, feelings of worthlessness, decreased interest in activities that were once pleasurable, difficulty in thinking or concentrating, continuous sadness, and thoughts of suicide or death. People with other illnesses such as heart disease, hormonal disorders, Parkinson’s disease, diabetes, and Alzheimer’s are also more likely to suffer from depression (Bjo¨rnsdo´ttir et al., 2016; Sundermann, 2016). Studies show that some patients experience both depression and anxiety (Ivanets et al., 2016). In addition, there seems to be a synergistic relationship between depression and anxiety (Beesdo et al., 2009; Kendler et al., 2007).

27.3.2 Epidemiology of depression and anxiety Depression leads to potential life lost due to disease burden, accounting for 4.4% of the total disability-adjusted life years in 2000 (Meyer, 2004). Depression has also been associated with an increased risk of mortality by 1.8% (1.6%
2.1%) (Cuijpers and Smit, 2002). It is a major global health problem, and it is estimated that by 2020, depressionrelated diseases will be the second leading cause of disease burden worldwide (Akhondzadeh-Basti et al., 2007;

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Akhondzadeh et al., 2005). The prevalence of depressive episode is estimated 11.3% among adult populations. The risk of depression in women is almost twice as much as men. It is estimated that the overall prevalence of depression in developed countries is 21% (Akhondzadeh et al., 2005). In addition, anxiety disorders are the most common class of psychiatric disorders (McLean et al., 2011). Anxiety is the response of the brain to the risks that stimulates the organism to deal with stimulus (Beesdo et al., 2009). Anxiety disorders are very common and the 12-month prevalence rate of these types of disorders is estimated to be 25% (Hettema et al., 2001).

27.3.3 Causes of depression and anxiety In neurochemistry, according to the amine hypothesis brain amines, especially norepinephrine and serotonin, are neurotransmitters that play a major role in the pathways associated with mood and behavior. Based on this hypothesis, a decrease in the activity of such amines may lead to depression. This hypothesis suggests that certain drugs can improve the activity of neurotransmitters such as serotonin and norepinephrine in the CNS and thereby improve the symptoms of depressive disorders (Baldessarini, 1975; Mendels and Frazer, 1974). Serotonin, which is secreted by the neurons of the digestive system and the CNS, is a chemical neurotransmitter that plays a role in transmitting neural signals. Low level of serotonin has been found to be the main cause of mood disorders and is a natural euphoric agent (Pradhan et al., 2014; Snyder, 2017). It is estimated that about 40 million brain cells are directly or indirectly affected by serotonin. These cells are associated with mood, libido, appetite, sleep, memory, learning, body temperature, and some social behaviors (Donaldson et al., 2014; Hostinar et al., 2014). Studies have shown that serotonin deficiency may affect mood and behavior and lead to depression, obsession, anxiety, panic, and excessive anger. Most antidepressants act through an increase serotonin levels in the blood or by upregulating serotonin receptors (Wohleb et al., 2016; Zhou et al., 2016). Serotonin levels are higher in men than in women, and although this difference is negligible, women are more likely to develop depression. There are several approaches to increase serotonin levels, including the use of vitamins, proteins, carbohydrates, and colored foods. Findings have shown that natural food coloring, or color additives such as saffron that contain yellow compounds (crocetin derivatives), can increase serotonin production in the brain (Young, 2007). Psychoanalytic theories suggest that internal psychological events and impulsive behavior are the determinant factors of anxiety. It has also been shown that environmental stress increases anxiety (Wohleb et al., 2016; Zhou et al., 2016). Cognitive theories suggest that events and problems are not the cause of anxiety and stress but that the interpretation of these events may lead to these problems.

27.4

Antidepressants

Medication therapy is one of the main therapeutic strategies for the treatment of depression, particularly in severe cases and in situations where a prompt response is required. Antidepressants are drugs that are used to treat acute depression and anxiety disorders. These drugs are not usually addictive if they are taken according to the physician’s instructions; however, there are some evidence that long-term use of antidepressants can cause withdrawal symptoms and physical dependence (Hjorth, 2016; Jin et al., 2016). They are also prescribed to manage various conditions, such as chronic pain, panic, agitation, and other mood disorders. Antidepressants were first introduced in the 1950s, when opioids were generally used as antidepressants. It may take several weeks to several years to feel the full therapeutic effects of an antidepressant. Antidepressants have also side effects along with the therapeutic potentials, and of course, the side effects do not appear in everyone. Therefore, the choice of medication is based on the physical condition, temperament, and lifestyle of the patient, and due to their long-term use, the potential side effects should also be considered.

27.4.1 Classification of antidepressants and antianxiety drugs Currently, 26 different drugs are approved for the treatment of depression by the US Food and Drug Administration (FDA). Based on the mechanistic pathway, antidepressants are classified into three subcategories including: MAOIs, amine reuptake inhibitors, and alpha 2 adrenergic receptor blocking agents. Amine reuptake inhibitors include two nonselective subgroups and SSRIs. In addition, the subcategory of nonselective drugs includes TCAs and heterocyclic ´ lamo, 2016). SSRIs are considered as the first-line treatment for depression in the elderly agents (Lo´pez-Mun˜oz and A (Alexopoulos, 2011).

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27.4.2 Pharmacokinetics Antidepressants pass through the bloodstream to reach the brain. Oral medications are dissolved and absorbed into the blood, depending on the fat solubility, local pH of the gastrointestinal tract, mobility, and the rate and extent of absorption. Antianxiety and antidepressant drugs, especially TCAs, are fat-soluble and have incomplete absorption. These drugs have higher rates of first-pass metabolism, and therefore reduced bioavailability. In addition, some antidepressants such as TCAs and SSRIs have active metabolites with long elimination half-lives (Spina et al., 2016).

27.4.3 Mechanism of action of antidepressants and antianxiety drugs Neural messages in the nervous system are transmitted through neurotransmitters, and a large number of neurotransmitters are released to exchange these messages. Synthesis, release, and reuptake of neurotransmitters allow the nervous system to reuse the neurotransmitters in later signal transduction processes. Antidepressants acts by increasing levels of neurotransmitters such as serotonin, norepinephrine, and dopamine in brain synapses. These neurotransmitters that are generally known as monoamine have critical role in thoughts, emotions, memory, and the regulation of some body functions. Any abnormal decrease in the levels of these chemical messengers can lead to depression. The level of monoamines can increase by blocking the receptors that enable neurotransmission (receptor blockers) or preventing the reabsorption of neurotransmitters (reuptake inhibitors) (Garcia-Miralles et al., 2016; Ji et al., 2014; Khan et al., 2016). All antidepressants have a similar effect on depression, although different patients may be more likely to respond to specific drugs than other patients. The choice of which antidepressant to use depends on several factors, including the symptoms of the patient, the side effects of a particular drug, and other disorders the patient may have. Antidepressants may be prescribed in combination with other medicines to treat depression symptoms. The effects of antidepressants on the brain and nerve signaling may not be immediate, and the response to drug therapy may take several weeks. Moreover, side effects such as nausea, insomnia, dryness of the tongue, and fatigue may appear at the beginning of treatment prior to therapeutic effects. In most cases, side effects improve within a few weeks. For better therapeutic outcomes, these medications should be taken for at least 6 months to reduce the risk of disease remission (Juurlink, 2016; Ji et al., 2014; Kawamura et al., 2017; Wang et al., 2010).

27.4.4 Possible side effects of antidepressants The side effects of antidepressants are often temporary and depend on dose. The FDA has warned that antidepressants can increase the risk of suicide in certain individuals. It is suggested that anyone who takes antidepressants be moni´ lamo, 2016). Side effects often occur during the early tored for unusual behavior (Gilron, 2016; Lo´pez-Mun˜oz and A stages of treatment with antidepressants and include the following: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Nausea: Decreases when taken with food. Weight gain: This side effect is more likely to occur with TCAs, and can be reduced with healthy diet and exercise. Fatigue: Taking medication before bed can reduce this side effect. Insomnia: This side effect is more likely to occur with the use of SSRIs, which can be controlled by taking drug in the morning. Dry mouth: Sipping water, sucking ice, and chewing gum can help to relieve this side effect. Constipation: This side effect is more likely to occur with TCAs, which can be reduced with fiber containing foods. Dizziness: Taking medication before bed can help to reduce this side effect. Restlessness: This side effect is most commonly associated with the use of SSRIs, and exercise can help to reduce this consequence. Sexual dysfunction: This side effect that is typically associated with the use of SSRIs includes the loss of libido and difficulty in sexual activity and orgasm. Replacing the drug with other antidepressant that is not related to sexual dysfunction, such as bupropion, nefazodone, or mirtazapine, or serotonin-norepinephrine reuptake inhibitors (SNRIs) can help to manage this side effect.

If antidepressants are abruptly discontinued, the patient may suffer withdrawal symptoms. These complications usually disappear after the drug leaves the patient’s body or after adjustment of the body to change the medication. Side effects of withdrawal are almost the same as side effects of drug therapy, in addition to headaches, irritability, diarrhea, and burning sensation. Although serotonin syndrome is a rare occurrence, it is a serious complication of any type of drug that increases serotonin levels, which should be diagnosed at early stages. This syndrome is often due to drug

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interactions (e.g., simultaneous use of MAOIs or TCAs and SSRIs). This situation may ultimately lead to muscle loss and liver failure. Initial symptoms of serotonin syndrome include confusion and blushing of the face (Hieronymus et al., 2016). SNRIs may increase blood pressure and heart rate and cause dizziness in rare cases (Jirkof, 2017; Ko¨hler et al., 2016; Procyshyn et al., 2017). Generally, fewer risks are associated with overdose of newer antidepressants in comparison with older drugs. In most cases, complications associated with overdose of these drugs are similar to the reported side effects of the drugs. TCAs can be very dangerous if they are consumed excessively and can possibly lead to death. Complications associated with overdose of antidepressants include fast and irregular heartbeat (erythema), high blood pressure, seizure, dry mucosa, dry and tender skin, mydriasis, restlessness, and hallucinations (Owens, 2014; Procyshyn et al., 2017).

27.5

Traditional medicine for the treatment of depression and anxiety

Many pharmaceutical studies have shown that some medicinal plants with psychotropic effects have much fewer side effects than antidepressants, and in some cases may have better therapeutic efficiency. Thus, the use of these plants may be considered as an alternative approach to the treatment of depression and anxiety (Akhondzadeh-Basti et al., 2007; Akhondzadeh et al., 2005). Although drug therapy and psychological interventions are the first-line approaches for the treatment of anxiety, herbal therapy may provide a safer and more effective option (Sarris et al., 2013; Sepahi et al., 2014). For example, medicinal plants such as Passiflora incarnata, commonly known as maypop, purple passionflower, true passionflower, wild apricot, and wild passion vine, have antianxiety properties (Vazirian et al., 2001). One study showed that P. incarnata has therapeutic effects on anxiety disorder, based on measurement of the severity of symptoms using Hamilton’s measurement scale for anxiety, which showed that the effects of 45 drops per day of this plant for four weeks is comparable to Oxazepam in reducing anxiety symptoms. Medicinal plants may also be a safe and effective alternative treatment for anxiety, depression, and related disorders (Akhondzadeh et al., 2004; Khorasany and Hosseinzadeh, 2016). Clinical studies on Echium amoenum and Silybum marianum extract showed that these plants are effective in the treatment of various types of depression such as obsessive-compulsive disorder (OCD) (Sayyah et al., 2009, 2010). Similar studies on the root extract of Withania samniferaand and Valeriana officinalis have shown positive effects on OCD (Jahanbakhsh et al., 2016). Many herbal remedies have comparable therapeutic effects and cause fewer side effects than drugs (Akhondzadeh et al., 2004; Kianbakht, 2008; Liakopoulou-Kyriakides and Kyriakidis, 2002). Saffron, in particular, has been shown to have satisfactory antidepression and antianxiety properties (Kianbakht, 2008).

27.6

Saffron

Saffron (Crocus sativus L.) is a plant of the Iridaceae family; it mainly contains water (10%
12%), minerals (5%
7%), a small amount of carbohydrates, wax (5%
8%), protein (12%
13%), and a small amount of essential oil, pigments, and flavonoids (Kianbakht, 2008; Nair et al., 1995). Other i components of saffron, which are also pharmaceutically active, include picrocrocin, safranal, and coloring agents such as carotene and crocin (Kianbakht, 2008).

27.6.1 Chemical compounds of saffron The bitter taste of saffron is attributed to a glycoside called picrocrocin, which is a colorless meothropen-aldehyde. This material is converted into aromatic aldehydes called safranal during thermal decomposition of the plant or enzymatic degradation. Safranal (C10H14O) is the essential oil of saffron and is responsible for the smell and aroma of this plant, which is produced by the separation of sugars from picrocrocin. Depending on the method used for drying, the concentration of safranal may vary after harvesting the plant. Crocin, a glycoside-containing carotenoid called crocetin and sugars, is responsible for the color of saffron. Other carotenoids, such as beta-carotene, lycopene, and zeaxanthin, and vitamins, especially riboflavin and thiamine, are also found in saffron. However, crocin, crocetin, and safranal are the main active ingredients of saffron (Liakopoulou-Kyriakides and Kyriakidis, 2002). The main coloring agent of saffron is attributed to the crocin. Crocin is a glucosidic derivative of crocetin. Accordingly, crocins have different types, with the highest concentration belonging to the digentiobiosyl ester of crocetin.

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27.6.2 Pharmaceutical applications of saffron in traditional medicine In traditional medicine, saffron has many therapeutic uses as it is used as a facilitator of digestion, appetizer, sedative, diaphoretic, mucolytic, stimulant, and aphrodisiac. It is also used for abortion and for the treatment of menstrual disorders, liver and gallbladder disorders, spasm, cramp, inflammation of the nasal and throat mucosa (Catarrh), insomnia, depression, cognitive disorders, and seizure. Saffron in Ayurveda medicine (traditional medicine in India) is used as an adjuvant for increasing body resistance against stress, trauma, anxiety, and fatigue (Akhondzadeh et al., 2005; Kianbakht and Ghazavi, 2005; Verma and Bordia, 1998). It has been shown that the saffron plant has many pharmacological properties including antiobstructive, antiischemic, antigenotoxic, anti-Alzheimer’s, anticoagulant, antiinflammatory, and antioxidant effects. Saffron also has protective effects against atherosclerosis and cardiovascular disease, diabetes, Parkinson’s disease, depression, and cancer and tumor activity (Abdullaev and Espinosa-Aguirre, 2004; Hosseinzadeh and Sadeghnia, 2007a; Hosseinzadeh and Ziaei, 2006). Although saffron and its active compounds have been shown to be effective in the treatment of painful menstruation, severe postpartum bleeding, and chronic bleeding from the uterus, as well as cardiovascular disorders and cancer, the main known therapeutic effects of saffron include treatment of insomnia, depression, cognitive disorders, and seizure (Abdullaev and Espinosa-Aguirre, 2004; Hosseinzadeh and Sadeghnia, 2007b; Hosseinzadeh et al., 2005, 2009; Xi et al., 2007).

27.6.3 Pharmacology of saffron and its active ingredients Different biological activities of saffron and its constituents have long been known (Hosseinzadeh and Sadeghnia, 2007b; Hosseinzadeh et al., 2005, 2009; Xi et al., 2007). It has been shown that following oral administration, crocin is hydrolyzed to croctein in the intestine, and then croctein is absorbed into the blood. However, crocin is not found in plasma, and it is mostly excreted in the intestine. Also, croctein does not accumulate in plasma by repeated oral administration of crocin (Giaccio, 2004). Saffron is known mainly for its exhilarating and antidepressant effects. Studies have shown the positive effects of saffron and saffron petal extracts in the treatment of mild-to-moderate depression (Christodoulou et al., 2015; Hausenblas et al., 2013). The medicinal properties of saffron may be attributed to a number of its constituents, such as crocetin, crocin, and safranal, which due to their strong antioxidant properties protect against reactive oxygen species and inflammatory cytokines by scavengering the free radicals. Nevertheless, a specific component of saffron that affects mood patterns and improves depressive symptoms has not yet been identified (Hausenblas et al., 2013; Hosseinzadeh and Jahanian, 2010; Rezaee and Hosseinzadeh, 2013). The effect of saffron is similar to that of diazepam, and like diazepam, as a benzodiazepine, it has an anxiolytic, analgesic, and relaxant effect (Azhari et al., 2014). A study on Wistar rats showed that the use of crocin for 3 weeks decreased the inactivity time in these animals in the swimming test. On the other hand, cAMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) levels increased at higher doses of crocin ($50 mg kg21), while VGF levels increased at all doses of crocin (12.5, 25, and 50 mg kg21) (Vahdati-Hassani et al., 2014). Saffron also increased the protein levels of BDNF and CREB, and transcription levels of BDNF (Ghasemi et al., 2015).

27.6.4 In vivo studies on the effects of saffron and its active compounds on depression and anxiety In vivo studies indicate that peritoneal injection of crocin, safranal, and aqueous and ethanolic extracts of saffron as well as saffron petal extract have antidepressant effects in mice (Rios et al., 1996). These studies showed that saffron and saffron petal extract, crocin, and saffronal increased climbing time and stereotypic activity. According to these results, crocin and safranal may be responsible for the antidepressant effect of saffron extract (Karimi et al., 2001; Rios et al., 1996). Intraperitoneal injection of kaempferol, a substance in saffron petals, also demonstrated an antidepressant effect (Hosseinzadeh et al., 2007). The use of 50, 100, and 200 mg of kaempferol per kg of body weight compared to fluoxetine has been shown to reduce symptoms of depression (Melnyk et al., 2010). Similarly, a comparison between the therapeutic potency of saffron and its active compounds (crocin and safranal) with fluoxetine and imipramine in the treatment of depression showed that crocin at different doses and safranal at 0.5 mg kg21 can improve depressive symptoms. Animal studies have also shown that alcoholic extract of saffron with doses of 200 and 800 mg and aqueous extract of saffron at doses of 160 and 320 mg kg21 of mice, compared to normal saline, decreased inactive time and improved the performance of rats compared to fluoxetine (Hosseinzadeh et al., 2003). Other findings have shown that saffron increases serotonin levels in the brain, but the exact mechanism is unknown. However, the results suggest that saffron may inhibit serotonin reuptake in the synapse (Wang et al., 2010). In addition,

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it has been shown that saffron in rats improves learning and spatial memory by influencing receptors involved in learning and spatial memory in nerve cells (Ghadami et al., 2007). The effects of aqueous or ethanolic extracts of C. sativus stigmas and safranal in the CNS have been widely studied and various pharmacological effects have been reported. Findings have shown that safranal and stigmas of the saffron have anticonvulsant and memory-improving effects. In addition, spatial cognitive abilities and contraction syndrome (withdrawal syndrome reduction) may also be improved by saffron extract (Hosseinzadeh and Jahanian, 2010; Hosseinzadeh and Ziaei, 2006; Rezaee and Hosseinzadeh, 2013). Moreover, a study on the use of saffron in rodents with depression showed that the aqueous-alcoholic extract of this plant improved depression. Also, crocin (50
600 mg kg21) reduced immobilization time and increased climbing time (Khazdair et al., 2015). Animal studies have also shown that the leaves and stigmas of saffron have antidepressant effects in mice. These findings confirmed that the antidepressant effects of saffron can be attributed to its major components, safranal and crocin (Verma and Bordia, 1998). Based on animal studies, it appears that crocin with tis antioxidant properties can affect serotonergic receptors and may also be effective in improving symptoms of OCD (Georgiadou et al., 2012). Intraperitoneal injection of crocin at a dose of 50 mg kg21 showed that the anxiolytic effect of crocin in rats was comparable to diazepam (Pitsikas et al., 2008). Also, evaluation of sleep activity and anxiety in mice showed that saffron at doses of 56 and 80 mg kg21 caused a significant reduction in anxiety and at a dose of 560 mg kg21 improved sleep. Studies have also demonstrated that safranal had antianxiety effects at doses of 0.15 and 0.35 mg kg21. These results showed that saffron and its components have different effects individually; however, these effects can be increased synergistically (Hosseinzadeh and Noraei, 2009; Pitsikas et al., 2008). In general, saffron seems to have beneficial effects on anxiety and insomnia in animal models, and these effects are dose-dependent (Hosseinzadeh et al., 2004). The effect of saffron and its components has been studied on various types of mental disorders including OCD in animal models. Considering that intense grooming behavior in animals is similar to those of OCD in humans, mCPP (serotonin agonist m-chlorophenylpiperazine) is known to induce self-grooming behavior in rats and exacerbate OCD symptoms. One study of the effectiveness of crocin on the animal model of OCD disorder indicated that crocin may reduce the 5HT-2c induced by mCPP through antagonizing the self-grooming receptor (Georgiadou et al., 2012). Other studies have shown the therapeutic effect of saffron on CNS disorders, particularly dementia and depression (Basti et al., 2007; Sarris et al., 2011). In general, saffron seems to have beneficial effects on anxiety and insomnia in animal models. These effects are dose-dependent and thus the holistic effects of saffron are more than its individual compounds (Nair et al., 1995).

27.6.5 Antidepressant and anxiolytic properties of saffron in clinical practice Various clinical studies have been conducted to evaluate the effects of saffron and its compounds in reducing various types of depression. The results of some double-blind studies on the therapeutic effects of saffron extract showed that saffron significantly improved mood in depressed and anxious people. Daily administration of saffron for 6 weeks was effective in treating mild-to-moderate depression (Akhondzadeh et al., 2004, 2005; Basti et al., 2007). Saffron extract has also been shown to reduce depression symptoms in 8 weeks of treatment (Moshiri et al., 2006). Moreover, comparison between the therapeutic effects of saffron and fluoxetine in patients with moderate depression showed that saffron was more effective than fluoxetine in treatment of mild-to-moderate depression (Moosavi et al., 2014). A similar study showed that daily intake of saffron for 6 weeks had a better effect on depression than fluoxetine in people with major depression (Marangoni et al., 2013; Shahmansouri et al., 2014). Also, comparison of the effect of saffron with imipramine in 40 patients with mild-to-moderate depression during 6 weeks of therapy showed that the therapeutic effect of saffron was comparable to imipramine; no significant difference in behavioral symptoms was observed between the two groups (Akhondzadeh et al., 2004). Another study on the effect of saffron on different types of depression showed that in women with premenstrual syndrome, consumption of saffron for 8 weeks reduced depression symptoms (Agha-Hosseini et al., 2008). Also, 1.4 mg powder of dried extract of saffron has been shown to effectively reduce the fatigue and anxiety of women during and after childbirth (Ahmadi et al., 2015; Shadipour et al., 2014). In addition, the results of a study on the effect of saffron and its compounds on different types of depression showed that saffron enhanced sexual activity in patients with depression (Modabbernia et al., 2012; Mansoori et al., 2011). Similarly, the results showed that coadministration of saffron with fluoxetine in women with depression and sexual dysfunction significantly improved symptoms including pain and irritability (Kashani et al., 2013).

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Regarding the prevalence of postpartum depression and also the acceptance of lactating women with depression from drugs with fewer side effects, effectiveness of saffron was evaluated in these patients and the results showed that saffron has favorable outcomes in these patients (Veysani and Miri, 2011). It was also shown that saffron has a wide therapeutic index and daily intake of 30 mg of saffron for 6
8 weeks is effective treatment for depression (Ulbricht et al., 2011). The results of a research on acute and subacute toxicity of saffron showed that saffron is a safe spice; however, lactating women should avoid taking high doses (over 750 mg kg21) of saffron (Bahmani et al., 2014). The results of a study on the effects of saffron showed that both the petals and stigma of saffron are effective in mild-to-moderate depression; both reduced the Hamilton Depression Scale (HAM-D) scale during 6 weeks of therapy (Basti et al., 2007). Also, clinical studies on the effect of saffron and its major compounds, including crocin, on depression and anxiety showed that the use of crocin for 4 weeks significantly improved the symptoms of depression and anxiety in patients compared to placebo group (Talaei et al., 2015). The results of a randomized study in patients with mild-to-moderate depression showed that hydroalcoholic extract of saffron stigma decreases the depression, anxiety, and depression/anxiety scores in patients with type 2 diabetes. Therefore, it was suggested that saffron, due to its negligible side effects, can be considered as an alternative treatment for the depression and anxiety associated with diabetes (Milajerdi et al., 2016; Taheri et al., 2014; Tsenkova et al., 2012). In sum, all clinical findings indicate that saffron and its components are likely to be effective in treating mild-tomoderate depression as much as the commonly used antidepressants. The antidepressant and anxiolytic properties of saffron in clinical use are summarized in Table 27.1. These effects may be related to the antiinflammatory and antioxidant properties of saffron, since it has been shown that using saffron in combination with curcumin, a substance in turmeric (Curcuma longa), can reduce depression and anxiety symptoms in people with major depression (Lopresti and Drummond, 2017).

27.6.6 Mechanistic pathway for antidepressant and antianxiety effects Saffron and saffron extract have been reported to increase serotonin levels in the brain. Although its mechanism is unknown, saffron may inhibit serotonin reuptake in synapses (Kianbakht and Ghazavi, 2005; Verma and Bordia, 1998). Further studies on animal models have shown that inhibition of aspartate reuptake and improvement of methyl monoamines and agonist of BDNF signaling can also be considered as effective mechanisms (Berger et al., 2011; Georgiadou et al., 2012; Wang et al., 2010). Serotonin reuptake inhibition in the synaptic terminals prolongs the half-life of serotonin throughout the brain and CNS and thus confronts the symptoms of depression. Therefore, the suggested mechanistic pathway for antidepressant effects of active constitutions of saffron (safranal and crocin) are mainly through the inhibition of serotonin reuptake, and the inhibition of dopamine and norepinephrine reuptake, which are supported by animal studies (Akhondzadeh et al., 2004; Kianbakht and Ghazavi, 2005; Verma and Bordia, 1998). The suggested mechanistic pathways for the antidepressant effects of safranal and crocin are summarized in Fig. 27.1. On the other hand, further studies have shown that saffron and its components, like other flavonoids, may exert their anxiolytic activity by interaction with the binding site of benzodiazepine in a GABA-A receptor. However, no proven mechanistic pathway for potential anxiolytic effects of saffron and its components have yet been determined. Evidence suggests that stress can trigger the hypothalamic-pituitary-adrenal (HPA) axis, leading to increased plasma cortisol levels as a response. Animal studies have shown that mice receiving saffron extract or crocin do not show an increase in stress-induced plasma corticosterone levels. Therefore, it is suggested that crocin may interact with the HPA axis and reduce corticosterone-induced stress. The results indicate that saffron inhibits n-methyl D-aspartate (NMDA) and sigma (σ) opioids receptors. The importance of this inhibition is that the sigma and NMDA receptors can regulate the release of corticosteroids from the cortex of the rat brain. These results obviously show that saffron and crocin inhibit corticosterone secretion in stressed mice by controlling the sigma and/or NMDA opioid receptors located on the adrenal cortex (Beesdo et al., 2009; Fava et al., 1997; Ivanets et al., 2016; Kendler et al., 2007; Kim et al., 2016a).

27.6.7 Effect of saffron and saffron compounds on nervous system diseases Saffron and its components, due to their high antioxidant properties, affect some of the processes in the nervous system, including memory disorders or weakening of nerves in Parkinson’s and Alzheimer’s disease. Based on the findings, saffron can prevent damage to brain cells by protecting the lipids around the cells. In multiple sclerosis, which is a demyelinating disease, this protective insulation of the lipids surrounding the brain cells is heterogeneously destroyed due to swelling of the supportive cells. Findings have shown that saffron and crocin can significantly reduce the swelling and cellular pressure that can lead to demyelination of nerve cells and atherosclerosis in the brain (Hausenblas et al., 2013).

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TABLE 27.1 Clinical trials on the antidepressant and anxiolytic effect of saffron and crocin. Results

Type of study

Component

Dose

Disorder

Type of control

References

Both groups improvement in mean HAM-D scores

Doubleblind active control randomized trial

Dried saffron stigma/ethanol extraction

30 mg day21

Depression

Imipramine (100 mg day21)

Akhondzadeh et al. (2004)

Both groups improvement in mean HAM-D scores

Doubleblind active control randomized trial

Dried saffron stigma/ethanol extraction

30 mg day21

Depression

Fluoxetine 20 (mg day21)

Noorbala et al. (2005)

Mean HAM-D scores for the saffron group lower at week 6, compared to placebo

Doubleblind placebocontrolled randomized trial

Dried saffron stigma/ethanol extraction

30 mg day21

Depression

Placebo

Akhondzadeh et al. (2005)

Improvement in mean HAM-D scores in saffron group by week two, compared to placebo

Doubleblind placebocontrolled randomized trial

Dried saffron petal/ethanol extraction

30 mg day21

Depression

Placebo

Moshiri et al. (2006)

Both groups improvement in mean HAM-D scores

Doubleblind active control randomized trial

Dried saffron petal/ethanol extraction

30 mg day21

Depression

Fluoxetine (20 mg day21)

Basti et al. (2007)

Curcumin and combined curcumin/saffron were effective in reducing depressive and anxiolytic symptoms

Doubleblind placebocontrolled randomized trial

Curcumin extract 1 saffron extract/ethanol extraction

Curcumin extract (250 mg) 1 15 mg saffron

Depressive and anxiety

Placebo

Lopresti and Drummond (2017)

Efficacy of 80 mg saffron daily in the treatment of mild-to-moderate depression (improvement in mean HAM-D scale)

Doubleblind active control randomized trial

Hydroalcoholic extract of saffron

40 and 80 mg day21

Depression

Fluoxetine (30 mg day21)

Moosavi et al. (2014)

Saffron can be as efficient as fluoxetine in improving depressive symptoms (HDRS scores)

Doubleblind active control randomized trial

Hydroalcoholic extract of saffron

30 mg day21

Depression

Fluoxetine (40 mg day21)

Shahmansouri et al. (2014)

Saffron shows attenuating effect on mild-to-moderate depression

Doubleblind placebocontrolled randomized trial

Hydroalcoholic extract of saffron

15 mg day21

Depression

Placebo

Modabbernia et al. (2012)

Significant improvement in scores of the HAM-D and HAM-A

Doubleblind active control randomized trial

Dried saffron petal/ethanol extraction

30 mg day21

Depression and anxiety

Citalopram (40 mg day21)

Ghajar et al. (2017)

(Continued )

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TABLE 27.1 (Continued) Results

Type of study

Component

Dose

Disorder

Type of control

References

Crocin in depression and could be administered in treatment of major depressive disorder patients

Doubleblind, placebocontrolled, pilot clinical trial

Crocin

Tablets (30 mg day21)

Depression and anxiety

Placebo

Talaei et al. (2015)

In all studies, saffron was given as a capsule, but crocin was prepared and taken as a pill.

FIGURE 27.1 Mechanistic pathways for the antidepressant effects of safranal and crocin.

Studies have also shown that saffron and crocin inhibit the accumulation of tau protein, an important factor in the occurrence of Alzheimer’s. It has also been shown that due to the effect of crocin in inhibiting apoptosis induced by beta-amyloid in brain cells (an effective source of Alzheimer’s disease), it can be considered as an effective drug in the prevention and control of Alzheimer’s disease (Hosseinzadeh and Jahanian, 2010; Rezaee and Hosseinzadeh, 2013). The results of a similar study showed that the use of aqueous extract of saffron (15 mg) and crocin (15 mg twice daily) had a positive effect on the treatment of patients with schizophrenia; in addition, no serious side effects were observed (Azhari et al., 2014).

27.7

Conclusion

Current therapeutic approaches for mood disorders such as depression and anxiety with chemical drugs such as fluoxetine, imipramine, mirtazapine, bupropion, etc., are associated with complications and side effects such as sexual dysfunction, insomnia, and weight gain. Hence, new treatment options with fewer side effects are important. Medicinal plants and herbal therapy are considered as safe and effective therapeutic options for treatment of mood disorders. Saffron and its active constituents safranal, crocin, and crocetin have been shown to have antidepressant and anxiolytic effects. Serotonin and dopamine reuptake inhibition are suggested as the most probable mechanism for saffron and crocin antidepressant effects. Animal studies as well as clinical trials have shown that the antidepressant effect of saffron extract and its active components, particularly crocin, is comparable and even higher than some conventional antidepressants. Therefore, findings suggest that saffron can not only treat depression and anxiety, but may also be helpful in the prevention of mood disorders.

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Chapter 28

Application of saffron as a neuroprotective agent Shahin Akhondzadeh, Seyyed-Hosein Mortazavi, Erfan Sahebolzamani and Amirhosein Mortezaei Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences, Tehran, Iran

Chapter Outline 28.1 Alzheimer’s disease 28.1.1 Introduction and epidemiology 28.1.2 Signs and symptoms 28.1.3 Pathogenesis 28.1.4 Pharmacological therapy 28.2 Saffron in Alzheimer’s disease treatment 28.2.1 Introduction to saffron

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28.2.2 Effects of saffron on the central nervous system 28.2.3 Neuroprotective activity of saffron 28.2.4 Saffron clinical trials on Alzheimer’s disease 28.3 Other herbal medicines and Alzheimer’s disease 28.4 Conclusion References

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Alzheimer’s disease

28.1.1 Introduction and epidemiology Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders known in the world. This condition usually presents with symptoms during old age. It is estimated that approximately 5 million people are living with AD in the United States and by 2050 this number is expected to exceed over 13.8 million Americans (Hebert et al., 2013; Kinney et al., 2018). Advancing age is one of the most important risk factors; thus, it is believed that after age 65, the risk for developing AD increases twofold every 5 years. Females are more likely to suffer from AD than males and in some studies family history of AD was reported (Jo¨nsson et al., 2006). AD has a significant financial burden on communities. According to some studies, the financial burden inflicted by AD is approximately $230 billion, a number that is expected to increase over $1.1 trillion by 2050 (Kinney et al., 2018). One study showed that there is a strong relationship between costs and cognitive changes (Jo¨nsson et al., 2006). In addition, patients with severe AD often depend on others and require extensive caregiver time (Herrmann and Gauthier, 2008).

28.1.2 Signs and symptoms Cognitive changes in AD follow a defined pattern. Its symptoms usually begin with memory problems. Subsequently, other brain functions are influenced and deterioration of virtually all intellectual functions, increasing apathy, and decreasing speech function gradually appear (Wolinsky et al., 2018). Tests are used to detect and measure memory and cognitive dysfunction such as the Alzheimer’s disease assessment scale cognitive subscale (ADAS-cog), clinical dementia rating scale sums of boxes (CDR-SOB), and mini-mental state examination (MMSE) (Hughes et al., 1982).

28.1.3 Pathogenesis Although there is no certain mechanism leading to Alzheimer’s, two theories are commonly presented as major explanations for the pathogenesis of AD (Kinney et al, 2018). The first mechanism is the presence of the misfolded amyloid Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00029-0 © 2020 Elsevier Inc. All rights reserved.

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plaques in the brain. When amyloid precursor protein is cleaved improperly by λ-secretase and erroneous β secretase, insoluble amyloid plaques aggregate in the brain. This theory arises from detection of certain gene mutations, which lead to significant increases in incidence of AD (Kinney et al., 2018; Wolinsky et al., 2018). The second important pathway leading to AD is hyperphosphorylation of tau, a protein that stabilizes microtubules in neurons. In addition, several studies have reported the significant role of uncontrolled brain inflammation behind AD progression. A number of examinations have shown high levels of inflammatory cytokines and macrophages in the histology of AD patient brains (Kinney et al., 2018). It is believed that in AD patients, the balance of neurotransmitter systems are impaired especially in glutaminergic and cholinergic systems. Excessive glutamate is released, which leads to cellular damage and cell death (Waite, 2015; Wolinsky et al., 2018).

28.1.4 Pharmacological therapy There are many pharmacological therapies to control cognitive dysfunction of AD; however, none are curative (Waite, 2015). Five drugs have been developed and used in several clinical trials: four acetylcholineesterase inhibitors (AChEIs) (donepezil, rivastigmine, galantamine, tacrine) and one N-methyl D-aspartate (NMDA) glutamate receptor blocker (memantine) (Waite, 2015; Wolinsky et al., 2018). Donepezil is approved to control mild-to-moderate AD. In one study, administration of donepezil over 6 months resulted in an improvement of 1.4 points on the MMSE (Waite, 2015). However, usually AChEIs are accompanied by nausea, vomiting, sleepless, weakness, and muscle cramps (Wolinsky et al., 2018). Memantine is another drug that has a significant effect on cognition and behavior and it is approved for moderate-to-severe Alzheimer’s (Herrmann and Gauthier, 2008). There are some alternative medications to alleviate symptoms of AD like antipsychotic drugs such as risperidone and antidepressant drugs such as citalopram, although they do not have a significant role in controlling symptoms (Wolinsky et al., 2018). Several new studies recommend novel strategies for AD therapy. One of the most important of these strategies is to decrease β amyloid aggregation in the brain by targeting the secretase enzyme (Waite, 2015).

28.2

Saffron in Alzheimer’s disease treatment

28.2.1 Introduction to saffron Herbal medicine still has an important role in primary health care for approximately 80% of the world’s population in both developing and developed countries due to better cultural acceptability and safety profiles (Akhondzadeh and Abbasi, 2006). Saffron is a spice derived from the flower of Crocus sativus. It is usually used as a nutritional supplement, and it is found all over the world. It is estimated that Iran supplies 90% of the world’s production of saffron (Christodoulou et al., 2015). Saffron includes many components like carbohydrates, polypeptides, lipids, and H2O; however, the main components with medical effects are crocetin, crocin, picrocrocin, and safranal (Khorasany and Hosseinzadeh, 2016). Saffron has many effects on the neurotransmitter network in the brain. Some limited investigations have been designed to assess the relationship between saffron and cholinergic activity. In one study, the inhibitory effect of aqueous methanolic saffron extract on AChE was reported (Geromichalos et al., 2012). Crocetin was found to bind to the catalytic and anionic site of AChE. This result demonstrated the potential effect of saffron to increase cholinergic activity. There have also been studies to assess the effects of saffron and its constituents on the glutamate system by focusing on NMDA receptors. The result of one study demonstrated that saffron extracts and crocetin had clear binding capacity at the phencyclidine binding site of the NMDA receptor and at the σ1 receptor (Hensel et al., 2008). The results also revealed that crocins and picrocrocin were not effective agonists. In an animal study, subchronic administration of ketamine, a noncompetitive NMDA receptor antagonist, caused impairment in cognitive function in rats and a single injection of crocins reversed recognition memory deficits produced by ketamine in rats (Georgiadou et al., 2014). It is believed that saffron can also increase reuptake inhibition of dopamine and norepinephrine (Alavizadeh and Hosseinzadeh, 2014).

28.2.2 Effects of saffron on the central nervous system Effects of saffron and its derivatives, like safranal, crocin, and crocetin, on the central nervous system (CNS) have been widely studied and various benefits have been elicited.

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Crocin, the active constituent of saffron, protects against beryllium chloride toxicity in rats through diminution of oxidative stress and increased gene expression of antioxidant enzymes (Lopresti and Drummond, 2014). It also attenuates lipid peroxidation and protects against tissue injury (Altinoz et al., 2016). Oral crocin may have protective effects against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats (Shafiee et al., 2018). In one study, it was shown that crocin can attenuate acute hypobaric hypoxia-induced cognitive deficits in rats (Zhang et al., 2018). Safranal is an active ingredient in saffron. Safranal attenuated cerebral ischemia induced oxidative damage in rat hippocampus. There is an antiabsence seizure property in safranal and its effect may be due to modifications on the benzodiazepine binding sites of the gamma-aminobutyric acid A (GABAA) receptor complex. Safranal can be effective in protecting a susceptible aged brain from oxidative damage by increasing antioxidant defenses (Samarghandian et al., 2016). Crocetin potentiates neurite growth in hippocampal neurons and facilitates functional (both motor and sensorimotor) recovery in rats with spinal cord injury (Wang et al, 2017a). Crocetin effectively attenuates neuropathic pain and significantly suppresses oxidative stress and neuroinflammation in the spared nerve injury (SNI) mouse model, supporting crocetin potential in treatment against neuropathic pain (Wang et al., 2017b). Crocetin attenuates spatial learning dysfunction and hippocampal injury in a model of vascular dementia (Lopresti and Drummond, 2014). Oxidative stress has an important role in progression of a number of neurodegenerative diseases, including AD. In several studies, a close relationship was observed between cognitive impairment and plasma antioxidant capacity of patients, and it is believed that progression of cognitive dysfunction is related to degree of oxidative stress (Perry et al., 2002). Studies have shown that saffron may play a role in attenuating age-related oxidative damage in the rat hippocampus (Samarghandian et al., 2016). The effect of saffron ethanol extract on inhibition of experimental autoimmune encephalomyelitis in C57bl/6 mice has also been studied and the results suggest for the first time that saffron is effective in prevention of symptomatic experimental autoimmune encephalomyelitis (EAE) by inhibition of oxidative stress and leukocyte infiltration to the CNS and may be potentially useful for the treatment of multiple sclerosis (Ghazavi et al., 2009). Crocin, one of the major component of saffron, was found as a potent neuronal antioxidant (Hosseinzadeh et al., 2009). In one study, crocin was reported as a unique and potent antioxidant in neurons, and it was observed that it can protect neuronally differentiated pheochromocytoma cells (PC-12) from peroxidation of their cell membrane lipids and decrease superoxide dismutase activity in the neurons (Ochiai et al., 2004). It has also been reported that crocin has a more antioxidant effect than α-tocopherol. In one animal study, there was observed that administration of crocin was effective to protect oligodendrocytes in the brain of the mouse (Nam et al., 2010). These studies confirm that crocin is an important agent to suppress oxidative stress and exhibits neuroprotective activities. The mechanism(s) underlying the effects of saffron and its active constituents on cognition is still a matter of investigation. Among the potential mechanisms, promotion of long-term potentiation, antiamyloidogenic activity, inhibition of AChE activity, and potent antioxidant properties are proposed to explain some of saffron effects on cognition. Longterm potentiation in the hippocampus is a form of activity-dependent synaptic plasticity and is believed to be a cellular basis for learning and memory. Reportedly, in this context, it was demonstrated that crocin promoted hippocampal long-term potentiation. In vitro enzymatic and molecular docking studies indicate that saffron and crocetin, but not safranal, inhibit AChE by binding to the catalytic center and the peripheral anionic sites. This AChE inhibitory activity displayed by saffron and crocetin promotes an increase in synaptic acetylcholine levels, a critical neurotransmitter in cognitive functions (Pitsikas, 2015). As mentioned previously, amyloid-β (Aβ) peptide and tau proteins play an important role in AD and new therapeutic strategies are focused on these targets. Aggregation of β amyloid can cause neuron cell apoptosis and synaptic dysfunction. Several studies have demonstrated inhibitory effects of saffron extract and crocin on fibrillogenesis of Aβ. One investigation revealed that saffron s extract tightens the blood
brain barrier and reduces amyloid β load and related toxicity in 5XFAD mice (Batarseh et al., 2017). One study exhibited inhibitory effects of saffron and crocin on fibrillogenesis of Aβ in a concentration and time-dependent manner (Papandreou et al., 2006); these results were also reported in other studies (Ghahghaei et al., 2012). Another investigation studied effects of crocin on fibrillogenesis of Aβ by thioflavine T-based fluorescence assay, DNA binding shift assay, CD spectroscope, and transmission electron microscopy. Its results showed that crocin, through stabilization of the helical structure, could prevent Aβ-mediated amyloid fibril formation in vitro (Ghahghaei et al., 2013). Some studies were designed to measure crocin’s neuroprotective activity against Aβ-induced toxicity in neuronal tissue (Morelli et al., 2016). Administration of Aβ produced significant damage to cells through induction of reactive oxidative species and apoptosis. The results showed that when Aβ was administered together with crocin, a significant dose-dependent inhibition of Aβ-induced apoptosis and reactive oxygen species production was observed. This may also suggest that crocin has the potential to prevent the aggregation of Aβ peptide and its adverse effects. Another important mechanism of AD is the abnormal accumulation of

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hyperphosphorylated tau protein and intracellular aggregation of microtubules-associated tau protein. It is believed that assembled microtubules and stabilization of the microtubule network plays an important role in signal transmission. Impaired phosphorylation of tau causes formation of neurofibrillary tangles, which leads to damaged neurons. Inhibition of abnormal hyperphosphorylation may provide a wide therapeutic target for AD (Iqbal et al., 2010). The inhibitory effects of crocin on aggregation of hyperphosphorylated tau was evaluated in one study using both biochemical methods and cell culture (Karakani et al., 2015). The results show that administration of crocin stabilizes the system by preventing fibrillation and lowering the tendency for aggregation, which was confirmed by transmission electron microscopy images. The results proved that crocin provided neuroprotection by reducing acrolein-induced Aβ and tau phosphorylation.

28.2.3 Neuroprotective activity of saffron Extensive studies have shown the impact of oxidative stress on the cognitive activity of neurons. Increased production of reactive oxygen species or reduced activity of antioxidant enzymes can lead to increased oxidative stress (Ghadrdoost et al., 2011). According to some studies, pathological situations such as stroke, trauma, or AD can deprive neurons of oxygen and lead to apoptosis. Oxidative stress is one of the mainstays in the process of cellular apoptosis and it appears that controlling it can be an important step in increasing the lifespan of neuronal cells (Soeda et al., 2016). The crucial point is the relevance of the level of these free radicals to the severity of cognitive impairments such as AD. In an animal study, crocin demonstrated its protective effect on oligodendrocytes of the mouse brain by suppressing the stress of the endoplasmic reticulum and improving the behavior of the animal (Deslauriers et al., 2011). In addition, the inhibitory effect of crocin on proapoptotic factors such as bcl-2 and Bax and Caspase-3 has been demonstrated in PC-12 cells damaged by H2O2 (Soeda et al., 2016). Crocin seems to be an effective agent in protecting the hippocampus. In an animal study, where streptozotocin induced diabetes on mice, administrating of crocin resulted in protection of the hippocampal neurons, improving learning activity and increasing antioxidant capacity (Tamaddonfard et al., 2013). In another study, the possible effect of saffron on the hypothalamus-pituitary-adrenal axis was debated and its protective role in improving cognitive activity was shown (Ghadrdoost et al., 2011). Saffron can have effective effects on motor activity. In an animal study where mouse dopaminergic neurons were eliminated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, administrating saffron reduced the damage to neurons by preserving neurotrophic factors such as brain-derived neurotrophic factor (BDNF) (Haeri et al., 2019). All of these studies have clearly shown the important role of saffron in protecting brain cells and point out that this substance can be a potential factor in the improvement of cognitive brain disorders.

28.2.4 Saffron clinical trials on Alzheimer’s disease Saffron was administrated in a few clinical trials and found to be comparable to AChEI drugs and NMDA receptor blockers. In a multicenter, randomized, double-blind controlled clinical study, saffron (30 mg day21 or 15 mg twice daily) or donepezil (10 mg day21) was administered to 54 patients aged 55 years and older with mild-to-moderate AD (Akhondzadeh et al., 2010b). The patients were followed over 22 weeks and were evaluated by ADAS-cog and CDRSOB. Saffron was found to be as effective as donepezil in this clinical trial. There was no significant difference between the two groups in frequency of side effects; however, patients in the saffron extract group experienced less vomiting. In another 16-week trial, saffron was compared with placebo (Akhondzadeh et al., 2010a). Forty-six patients with mild-to-moderate AD were randomly assigned to two groups to receive saffron (30 mg day21 or 15 mg per capsule, twice per day) or placebo (two capsules per day). Cognitive status and clinical profiles of patients were assessed by ADAS-cog and CDR-SOB. At the end of the trial, cognitive functions in the saffron-treated group were significantly improved compared to the placebo group and the frequency of adverse effects between the two groups was similar. There was also a trial that investigated the efficacy and safety of saffron for the treatment of severe Alzheimer’s. In a randomized, double-blind, parallel group trial, saffron was compared with memantine (Farokhnia et al., 2014). In this study, 68 patients with moderate-to-severe AD received memantine (20 mg day21) or saffron extract (30 mg day21) capsules for 12 months. Participants were evaluated every month by the severe cognitive impairment rating scale and functional assessment staging along with recording of probable adverse events. Results revealed that the efficacy of saffron was comparable with memantine in decreasing cognitive dysfunction in patients with moderate-to-severe AD. There were no significant differences in adverse effects between the two treatment groups.

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28.3

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Other herbal medicines and Alzheimer’s disease

The concept of herbal medicine is variable and includes simple herbal supplementary agents and therapeutic agents in the traditional medicinal system. Studies evaluating the efficacy of herbal medicine have found solid evidence for its use in the treatment of AD. In addition, research investigating the anti-AD effects of herbal medicine in experimental models are growing. Ginkgo biloba leaf, also called ginkgo and Panax ginseng root, commonly called ginseng, are the most famous and top-selling herbal supplements for their antioxidant and/or memory-enhancing properties. However, evaluation of their preventive or therapeutic effect in AD patients does not seem to have reached an end (Geun Kim and Sook Oh, 2012).

28.4

Conclusion

AD is one of the leading causes of dementia in the world and its progression involves many factors including cholinesterase activity and Aβ and tau proteins. Current therapies with AChE inhibitors and NMDA glutamate receptor blockers are only useful for delaying or preventing symptoms of AD for a limited time. Research targeting Aβ and tau proteins is limited and its efficacy in improving cognition has not been validated in human trials. Natural alternatives that exhibit beneficial effects via multiple targets and pathways could be valuable options. According to the studies presented in this chapter, saffron and particularly crocin have positive effects on the CNS, and crocin, in particular, appears to be multifunctional in protecting brain cells, modulating aggregation of Aβ and tau proteins, and attenuating cognitive and memory impairment. Although the number of saffron clinical studies in treatment of AD is limited, the results are promising. The findings from animal studies of crocin as well as the clinical studies of saffron confirm crocin as an effective natural alternative in the prevention and treatment of AD.

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Chapter 29

Cardiovascular effects of saffron and its active constituents Bibi-Marjan Razavi1 and Hossein Hosseinzadeh2 1

Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran,

2

Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 29.1 Introduction 451 29.2 Cardiovascular pharmacological effects of saffron 452 29.2.1 Antiarrhythmic and antiischemic effects 452 29.2.2 Protective effects against cardiac hypertrophy 453 29.2.3 Effects of saffron and its active constituents on blood pressure 453

29.1

29.2.4 Antiatherosclerotic effects of saffron and its active constituents 454 29.2.5 Protective effects of saffron and its active constituents on natural and chemical toxins 455 29.3 Conclusion 457 References 457

Introduction

Saffron (Crocus sativus L.) is used as a coloring and flavoring agent in food preparation as well as in perfumes and cosmetics (Alavizadeh and Hosseinzadeh, 2014; Mollazadeh et al., 2015). The main components of saffron stigmas are carotenoids (crocetin, crocins, α-carotene, lycopene, zeaxanthin), monoterpene aldehydes (picrocrocin and safranal), monoterpenoids (crocusatines), isophorones, and flavonoids. Crocins and crocetin are saffron coloring agents, while the unique aroma of saffron is related to safranal (Hosseinzadeh and Nassiri-Asl, 2013). Additionally, saffron has been employed for many purposes in traditional medicine, and therefore the pharmacological activities of saffron and its constituents have been extensively studied. These include antioxidant (Hosseinzadeh et al., 2009b), antinociceptive (Amin and Hosseinzadeh, 2012; Amin et al., 2017), antiinflammatory (Hosseinzadeh and Younesi, 2002), antidepressant (Ghasemi et al., 2015; Hosseinzadeh et al., 2004, 2007; Vahdati-Hassani et al., 2014), anxiolytic (Hosseinzadeh and Noraei, 2009), anticonvulsive (Sadeghnia et al., 2008), antitussive (Hosseinzadeh and Ghenaati, 2006), antiischemic (Hosseinzadeh et al., 2009a), anti-Alzheimer’s (Hosseinzadeh and Ziaei, 2006; Hosseinzadeh et al., 2012), antigenotoxic (Hosseinzadeh et al., 2007a), and anticancer activities (Rastgoo et al., 2013). Saffron also functions as an antidote to various toxic insults (Razavi and Hosseinzadeh, 2015) and exhibits hypolipidemic (Sheng et al., 2006) effects. Accumulating evidence supports protective effects of saffron and its active components in different organs such as the brain (Dorri et al., 2015; Kamyar et al., 2016; Mehri et al., 2012), heart (Razavi et al., 2013a), kidney (Amin et al., 2015), liver (Lari et al., 2014, 2015), gastrointestinal tract (Khorasany and Hosseinzadeh, 2016), and immune system (Khajuria et al., 2010). Saffron is also able to manage metabolic syndrome (Razavi and Hosseinzadeh, 2017). Because of its safety (Bostan et al., 2017), unique antioxidant and antiinflammatory properties, and ability to decrease lipid levels, saffron may be one of the best supplements for cardiac health. In Mediterranean countries the incidence of heart disease is lower than other countries, possibly due to the common use of saffron (Kamalipour and Akhondzadeh, 2011). Saffron is found to reduce the risk of cardiovascular disorders such as arrhythmia, ischemic reperfusion, hypertrophy, hypertension, and atherosclerosis (Fig. 29.1). In this chapter, we summarize various studies related to the effects of saffron and its main constituents on the cardiovascular system.

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00030-7 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 29.1 Schematic description of the effects of saffron and its main constituents on the cardiovascular system.

29.2

Cardiovascular pharmacological effects of saffron

29.2.1 Antiarrhythmic and antiischemic effects Antiarrhythmic effects of saffron have been identified in several studies. The effects of aqueous ethanolic extract of saffron on heart rate and contractility were evaluated on isolated guinea pig hearts. Significant reductions in heart rate and contractility were observed. The suppressive effects of the extract on guinea pig heart rate and contractility may be due to a potent inhibitory effect on calcium channels (Boskabady et al., 2008). In another study on isolated, perfused atrioventricular (AV) nodes of rabbits, saffron protected AV nodes from supraventricular arrhythmia. According to the results of this study, saffron nonspecifically affected the transitional cells of the fast nodal pathway through a rateindependent increase in basic and functional (facilitation and fatigue) parameters of the AV node (Khori et al., 2006.). In addition, the findings of another study suggested that saffron has no toxic effects on cardiac autonomic nervous system activity. Moreover, the stability of heart sympathovagal balance may be improved by saffron in normal rats (Joukar and Dehesh, 2015). Myocardial ischemia is defined as the reduction of blood flow to cardiac muscle as a result of partial or complete blockade of the coronary arteries. Although reperfusion is considered a recovery process for the ischemic myocardium, it often induces damage to the reperfused tissue because of the increased generation of reactive oxygen species (ROS) (Zhou et al., 2015). Saffron was found in several investigations to attenuate ischemia-reperfusion (IR) injuries because of its antioxidant effects. For instance, oral administration of saffron for 6 weeks improved left ventricle pressure, heart rate, coronary flow, and left ventricle end diastolic pressure. Results also indicated that saffron reduced infarct size, lowered lipid peroxidation, and increased glutathione peroxidase (GPX) activity. Furthermore, saffron restored the decreased level of phosphorylated Akt and 4EBP1 and reduced the level of p38 compared to IR hearts (Nader et al., 2016). Another study indicated that crocin [20 mg kg21 day21, intraperitoneally (IP), for 21 days] improved reperfusioninduced arrhythmias. Data showed IR injury significantly reduced superoxide dismutase (SOD) activity and glutathion (GSH) content and elevated malondealdehyde (MDA) levels in heart muscle. Crocin significantly increased catalase activity in heart tissue compared to the IR group due to its antioxidant activity. The use of crocin for treatment or prevention of arrhythmias in patients with ischemic heart disease was suggested in this study (Jahanbakhsh et al., 2012). In a similar study, treatment with saffron extract (100 mg kg21 day21, orally) for 7 days prior to IR injury reduced the susceptibility and occurrence of lethal ventricular arrhythmia during the reperfusion. This protection may be attributed to a decrease in electrical conductivity and prolonged duration of the action potential (Joukar et al., 2013). As cardiac IR is associated with oxidative injury, another study compared the protective effects of crocin (40 mg kg21, orally for 21 days) and vitamin E during IR. Results in an isolated rat heart model indicated that crocin exhibited the same protective effect as vitamin E against cardiac IR injury by elevation of total antioxidant capacity (Dianat et al., 2014a,b). In addition to crocin, safranal is also able to protect against IR injury. To demonstrate this, safranal (0.1
0.5 mL kg21 day21, IP) was administrated to rats for 14 days, and on day 15 one-stage ligation of the left anterior descending coronary artery was performed for 45 minutes followed by 60-minute reperfusion. Safranal reduced infarct size, improved left ventricular functions, and modulated hemodynamic heart parameters. The probable mechanism of safranal protection is increased phosphorylation of Akt/glycogen synthase kinase-3b/eNOS pathway and decreased IKK-b/NFκB protein expression in heart tissue. Moreover, safranal increased the levels of myocardial

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antioxidant and decreased the level of nitrotyrosine. The increased level of creatine kinase MB (CK-MB) and decreased level of lactate dehydrogenase (LDH) in IR heart, were normalized in safranal pretreated rats. Safranal reduced tumor necrosis factor-α (TNF-α) levels in IR heart in a dose-dependent manner. Safranal also protected myocardial architecture and reduced inflammatory cell numbers and edema (Bharti et al., 2011).

29.2.2 Protective effects against cardiac hypertrophy Cardiac hypertrophy, an independent risk factor of cardiovascular disease, is an important cause of morbidity and mortality worldwide. Prolonged compensatory adaptation of cardiac hypertrophy leads to worsening myocardium, both functionally and histologically. It has been suggested that cardiac hypertrophy and cardiac failure are induced by mechanical left ventricular wall stress as a result of induction of ROS (Das et al., 2004). It has been demonstrated that crocetin (25 and 50 mg kg21, IP, for 15 days) prevented cardiac hypertrophy induced by norephinephrine (NE) through inhibition of lipid peroxidation and increased the activity of antioxidant enzymes such as SOD and GPX. An additional study showed that crocetin significantly repaired myocardial damages induced by NE. Results indicated that the antioxidative effects of crocetin were stronger than those of captopril (the positive control); however, the effect of crocetin on improving cardiac hypertrophy, particularly on the left ventricular index, was less than captopril (Shen and Qian, 2006). Results of another study by Shen et al. (2006) showed crocetin increased both cardiac Na1 K1 ATPase and mitochondrial Ca21 Mg21 ATPase activity and significantly inhibited the activity of matrix metalloproteinase-2 (MMP-2) as well as MMP-2 and MMP-9 mRNA expression. In primary culture of cardiac myocytes exposed to noradrenaline, crocetin significantly reduced the activity of LDH and elevated mitochondrial succinic dehydrogenase activity, ATPase (Na1 K1 ATPase and Ca21 ATPase) activity, and mitochondrial membrane potential. Therefore crocetin suppressed the impairment of energy metabolism and attenuated the induction of apoptosis in cardiac myocytes exposed to noradrenaline (Shen et al., 2004). Another study showed that crocetin (1
10 μM) inhibited cardiac hypertrophy induced by angiotensin II (Ag II) in cultures of primary cardiac myocytes and fibroblasts in a dose-dependent manner. Moreover, crocetin (50 mg kg21 day21) protected and reversed cardiac hypertrophy induced by aortic banding in vivo. According to this study, crocetin not only prevented the development of cardiac hypertrophy but also reversed established cardiac hypertrophy by inhibiting hypertrophy, inflammation, and fibrosis dependent on ROS and the MEK-ERK1/2 kinase pathway. Thus the MEK-ERK1/2 pathway is a target of the inhibitory effects of crocetin (Cai et al., 2009). Furthermore, in a rat model of cardiac hypertrophy induced by overloading pressure, crocetin (50 and 100 mg kg21, gavage, for 30 days) reduced the cardiac indexes and hydroxyproline content in the heart, increased the activity of Na1 K1 ATPase and Ca21 Mg21 ATPase, as well as attenuated the activity of MMPs (Shen and Qian, 2004).

29.2.3 Effects of saffron and its active constituents on blood pressure Antihypertensive effects of saffron and its main constituents have been shown in both acute and chronic administration during both animal and clinical studies. Mechanisms including the blocking of calcium channels, inhibition of sarcoplasmic reticulum Ca21 release into the cytosol, interaction with endothelial nitric oxide (NO), and antioxidant activity may be involved in hypotension induced by saffron. According to the study by Razavi et al. (2016a), an endothelium independent mechanism may also be involved in vasodilatory and hypotensive effects of safranal. Aqueous and ethanolic extracts of saffron petals were shown to reduce mean arterial blood pressure (BP) in anaesthetized rats in a dose-dependent manner and to inhibit contractile responses produced by electrical field stimulation of isolated rat vas deferens and guinea pig ileum (Fatehi et al., 2003). In addition, dose-dependent hypotensive effects of intravenous injection of aqueous extract of saffron stigma (2.5, 5, and 10 mg kg21) and its two active constitutes have been demonstrated in normotensive and hypertensive anaesthetized rats. As reflex tachycardia was not observed in this study, it could be suggested that both heart function and blood vessel contractility are affected by saffron. The effect of safranal on lowering BP was greater than that of other saffron components. Crocin significantly reduced BP in hypertensive anaesthetized rats (Imenshahidi et al., 2010). Other studies indicated that chronic administration of saffron aqueous extract, crocin, or safranal could reduce the mean systolic BP in deoxycorticosterone acetate salt-treated rats. The results also showed that the antihypertensive effects of these agents did not persist (Imenshahidi et al., 2013, 2014, 2015). Oral treatment with hydroalcoholic extract of saffron (200 mg kg day21) for 5 weeks reduced BP in rats with hypertension induced by NG-nitro-L-arginine methyl ester in drinking water and decreased the cross section area, median thickness, and elastic lamellae number of the aorta (Nasiri et al., 2015). The vasomodulatory effects of crocetin in hypertension has been established in another study,

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FIGURE 29.2 Different mechanisms of antiatherosclerotic and antihypertensive effects of saffron. EC, Endothelial cell; ICAM, intercellular adhesion molecule; Ox-LDL, oxidized LDL; VCAM, vascular cell adhesion molecule; VSMCs, vascular smooth muscle cells.

which indicated crocetin improved endothelium-dependent acetylcholine relaxations through endothelial NO but not the cyclooxygenase pathway (Mancini et al., 2014) (Fig. 29.2). In a double-blind, placebo-controlled randomized study on 260 infertile men, treatment with saffron (60 mg day21 for 26 weeks) caused an 11.8% decrease in mean systolic BP and a 10.8% decrease in mean diastolic BP (Safarinejad et al., 2011). In another double-blind, placebo-controlled study in healthy adult volunteers, saffron tablets (400 mg day21 for 1 week) significantly reduced standing systolic BP and mean arterial pressure (Modaghegh et al., 2008).

29.2.4 Antiatherosclerotic effects of saffron and its active constituents Antiatherosclerotic effects of saffron and its main components have been evaluated in different studies. Due to antioxidant and inhibitory effects on endothelial cell apoptosis, atherosclerosis can be prevented by saffron. In vitro studies indicated that crocin increased intracellular calcium induced by H2O2 in bovine aortic endothelial cells (BAECs) due to antioxidant and antiapoptotic effects by downregulating the increased level of Bax/bcl2 (He et al., 2004; Xu et al., 2006). Endothelial dysfunction is involved in the initiation and progression of atherosclerosis. In one study, endothelial dysfunction was induced in both in vivo and in vitro experiments. In vivo, feeding of a high cholesterol diet to rabbits and in vitro treatment of BAECs with oxidized LDL (ox-LDL) were used. Results showed that crocetin significantly improved the endothelium-dependent relaxation of the thoracic aorta in hypercholesterolemic rabbits through increased aortic endothelial nitric oxide synthase (eNOS) activity, which led to elevation of NO production (Tang et al., 2006). In another experiment, crocetin protected advanced glycation end products (AGEs)-induced bovine endothelial cell apoptosis and adhesion of leukocytes to endothelial cells. This effect occurred through ROS inhibition, intracellular calcium stabilization, and downregulation of the expression of intercellular adhesion molecule-1 (ICAM-1), suggesting a beneficial effect of crocetin on prevention of diabetes-induced vascular complications (Xiang et al., 2006a,b). Similar results were observed during treatment with crocetin in human umbilical vein endothelial cells in which crocetin inhibited high glucose-induced apoptosis through the PI3K/Akt/eNOS pathway (Meng and Cui, 2008). In addition, the protective effects of crocetin against the migration and proliferation of vascular smooth muscle cells (VSMCs) induced by AGEs have been shown. Crocetin reduced the levels of TNF-α, IL-6, and MMP-2 and MMP-9. Considering these studies, it can be suggested that crocetin may have a beneficial effect in preventing diabetes-associated cardiovascular complications (Xiang et al., 2017). VSMC proliferation plays a main role in the development and progression of atherosclerosis. A study showed that crocetin alleviated angiotensin II-induced VSMC proliferation, potentially in part due to its inhibition of ERK1/2 through a calcium-dependent pathway (Zhou et al., 2006, 2007) and inhibition of PKC activity (Zhou et al., 2010b). Inhibition of the cell cycle G1/S transition by crocetin in VSMC is mediated through suppression of cyclin D1 and elevation of p27kip1 (Zhou et al., 2010a).

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Moreover, in another study crocin inhibited the proliferation of SMCs and formation of foam cells induced by oxLDL in bovine aortic smooth muscle cells (BASMCs) in a concentration-dependent manner, which promoted the initiation and progression of atherosclerosis. Crocin also inhibited total cholesterol (TC) and cholesteryl ester accumulation induced by ox-LDL in macrophages. Crocin could also inhibit intracellular calcium elevation in SMC. It is suggested that crocin showed antiatherosclerotic effects via decreasing the level of ox-LDL, which has an important role in the initiation and progression of atherosclerosis. In addition, in ox-LDL and hyperlipidemic diet-induced atherosclerosis in quails, crocin could reduce MDA and increase NO in serum, decrease the level of serum TC, triglyceride (TG), lowdensity lipoprotein-cholesterol (LDL-C), and inhibit the formation of aortic plaques (He et al., 2005, 2007). Furthermore, in another study suppression of LDL oxidation by crocetin attenuated atherosclerosis in hyperlipidemic rabbits (Zheng et al., 2006). The lipid lowering effect of saffron is considered another antiathersclerotic mechanism. Different studies indicated saffron and its active constituents modulated serum TC, TG, LDL, and high-density lipoprotein (HDL). For example, a mixture of extracts from stigma and petal of saffron improved dyslipidemia in obese rats and reduced atherosclerosis. The atherosclerosis index (LDL/HDL) and atherogenic index (TC/HDL) were also improved after saffron treatment (Hoshyar et al., 2016). Another study conducted on rats that received a high fat diet for 12 weeks indicated that crocin (80 mg kg21) significantly reduced plasma levels of TG and TC, whereas saffron ethanolic extract (40 mg kg21) significantly improved their atherogenic index (the level of LDL/HDL) (Mashmoul et al., 2014). Moreover, a 10-day treatment with crocin (25
100 mg kg21) significantly reduced TG, TC, LDL-C, and very low-density lipoproteincholesterol (VLDL-C) in diet-induced hyperlipidemic rats through inhibition of pancreatic lipase, which leads to the malabsorption of fat and cholesterol (Sheng et al., 2006). Besides crocin, crocetin (25 and 50 mg kg21 for 10 weeks) also reduced high cholesterol diet-induced dyslipidemia in rats, potentially due to antioxidant and antiinflammatory effects as well as downregulation of phosphorylated p38 MAPK (Diao et al., 2018). In another study of hypercholesterolemia-induced atherosclerosis in rabbits, crocetin reduced NF-κB activation, resulting in suppressed expression of the adhesion molecule VCAM-1 (Zheng et al., 2005). In addition to antihyperlipidemic effects, a hydromethanolic extract of saffron exhibited hypolipidemic effects in healthy male rats. The hydromethanolic saffron extract (50 mg kg21, IP) significantly reduced serum TC levels in healthy male rats after 14 days of treatment (Arasteh et al., 2010). It is well-known that adiponectin has an important role in the regulation of lipid metabolism. Reduced levels of adiponectin, a cytokine released from adipose tissue, are associated with hypertension, hyperlipidemia, diabetes, and atherosclerosis (Izadi et al., 2013). It was demonstrated that ethanolic and aqueous saffron extracts significantly increased adiponectin levels in streptozotocin (STZ) diabetic rats (Hemmati et al., 2015). It was also found that saffron can moderately stimulate peroxisome proliferator-activated receptor α (PPARα). PPARα activation has a role in lipid profile improvement and atherosclerosis (Duval et al., 2007). Thus another mechanism of saffron antiatherogenic effects could be PPARα activation. In a randomized, placebo-controlled clinical trial on patients with metabolic syndrome, the attenuating effect of saffron (100 mg day21) on serum heat shock protein (HSPs 27 and 70) antibody titers was demonstrated. Following exposure to stressful conditions such as several cardiovascular disease risk factors, the expression of HSPs was increased. According to the literature, there is a positive association between plasma antibody titers to HSPs and cardiovascular disease such as atherosclerosis (Shemshiana et al., 2014) (Fig. 29.2). Inhibitory effects of saffron on platelet aggregation and coagulation have been shown in multiple studies. Saffron aqueous extract inhibited platelet aggregation induced by ADP, epinephrine, and collagen in human platelets (Jessie and Krishnakantha, 2005). The presence of both platelet aggregation inducer and inhibitor has been identified in bulbs of C. sativus var. Cartwrightianus (Liakopoulou-Kyriakides and Skubas, 1990). In contrast to the work discussed earlier, saffron tablets (200 and 400 mg kg21) ingested for 1 week had no effect on coagulant and anticoagulant systems (Ayatollahi et al., 2014) (Fig. 29.3).

29.2.5 Protective effects of saffron and its active constituents on natural and chemical toxins The protective effects of saffron and its active constituents have been shown against some chemical and natural toxins including diazinon (an organophosphate insecticide), isoproterenol (a synthetic nonselective β adrenoceptor), doxorubicin (an antitumor agent), and patulin (a mycotoxin). It has been reported that diazinon (15 mg kg21, gavage, for 28 days) induced cardiovascular toxicity due to oxidative stress. In isolated rat aorta, crocin (20 mg kg21, IP, for 28 days) decreased toxic effects of diazinon through decreasing lipid peroxidation and improving impaired contractile and relaxant responses in rat aorta (Razavi et al., 2014). Moreover, the BP normalizing effect of crocin has been demonstrated in a study that indicated coadministration of crocin and diazinon restored the increase of systolic BP and decrease of heart

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FIGURE 29.3 Schematic description of antiatherosclerotic mechanisms of saffron and its main constituents. ICAM-1, Intercellular adhesion molecule-1; SMC, smooth muscle cell; VCAM-1, vascular cell adhesion molecule-1.

rate caused by subchronic diazinon administration in rats (Razavi et al., 2013b). In addition to antioxidant effects, other research has shown that diazinon-induced apoptosis by activation of caspase 9 and caspase 3 and via elevation of the Bax/Bcl2 ratio. Crocin inhibited apoptosis in aortic tissue (Razavi et al., 2016b). Data obtained from in vivo studies revealed that crocin protected against diazinon-induced oxidative stress and mitochondrial-mediated apoptosis in heart tissue of rats after subchronic exposure (Razavi et al., 2013a). The increased level of CK-MB (a cardiac injury biomarker) due to diazinon exposure in rats was reduced following treatment by crocin, saffron aqueous extract, and safranal (Hariri et al., 2014; Razavi et al., 2013a). Studies have evaluated the cardioprotective effect of extracts from saffron, crocin, and safranal in isoproterenolinduced myocardial infarction in rats. According to these studies, crocin (20 mg kg21 day21, IP, for 21 days), saffron extract (20, 40, 80, and 160 mg kg21, IP) and safranal (0.025, 0.050, and 0.075 mL kg21 IP) for 8 days, significantly reduced the LDH and CK-MB in serum and myocardial lipid peroxidation induced by isoproterenol. Moreover, histopathological examination showed that saffron and its active constituents restored myocardial injury induced by isoproterenol (Goyal et al., 2010; Mehdizadeh et al., 2013). In another study, oral administration of saffron (200, 400, and 800 mg kg21) for 4 weeks showed significant cardioprotective effects against isoproterenol-induced myocardial injury through stabilizing hemodynamics and left ventricular functions, restoring structural integrity, and enhancing antioxidant status. Saffron at 400 mg kg21 showed maximum protective effects (Sachdeva et al., 2012). In isolated rabbit hearts, ROS were generated by two models including electrolysis of the perfused heart solution and/or perfusion with 30 μM doxorubicin, both in the presence and absence of 10 μg mL21 saffron extracts. Results indicated that ROS decreased ventricular pressure, heart rate, and coronary flow and elevated lipid peroxidation, whereas SOD activity decreased. ROS also induced myocardial architecture alteration. Perfusion with saffron during electrolysis decreased ROS and improved myocardial function. However, the effect of saffron against doxorubicin was less suggesting that mechanisms other than oxidative stress may be involved in doxorubicin cardiotoxicity (Chahine et al., 2013). In another study conducted by the same authors, doxorubicin increased ischemic tissue damage in isolated rabbit heart during 40 minutes of reperfusion. Saffron extract significantly decreased oxidative myocardial damage during the first minutes of reperfusion, but the effect was less than when given before ischemia. Saffron increased cardiac troponin T proteins, inhibited the p38 mitogen-activated protein kinases pathway, and activated the AKT/mTOR (mammalian target of rapamycin)/4EBP1 pathway in reperfusion and doxorubicin treated rabbit hearts (Chahine et al., 2014). Crocin is able to protect the heart against toxicity induced by natural toxins such as patulin. Patulin is a mycotoxin produced principally by Penicillium expansum but also by several species of the genera of Penicillium, Aspergillus, and Byssochlamys. One study found that patulin induced cardiotoxicity by increasing the level of creatinin phosphokinase,

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elevation of lipid and protein oxidation, induction of apoptosis through overexpression of P53, and activation of caspase 3. Crocin improved all toxic cardiac effects of patulin in mice (Boussabbeh et al., 2015).

29.3

Conclusion

This chapter suggests that saffron and its constituents including crocin, crocetin, and safranal may be potential candidate medicines against CVDs. Different in vivo and in vitro studies regarding the beneficial effects of saffron in CVD including arrhythmia, IR, hypertrophy, hypertension, and atherosclerosis were introduced. Saffron was found to attenuate the deleterious effects on the cardiovascular system induced by natural and chemical toxins such as diazinon, doxorubicin, and patulin. Several mechanisms including antioxidant, antiapoptotic, hypolipidemic, antiinflammatory, vasodilator, and improvement of antioxidant defense systems are involved in cardiovascular protection induced by saffron. This chapter also suggests that after randomized clinical trials saffron may be considered as a preventive or therapeutic agent against CVDs.

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565. Vahdati-Hassani, F., Naseri, V., Razavi, B., Mehri, S., Abnous, K., Hosseinzadeh, H., 2014. Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus. DARU J. Pharm. Sci. 22 (1), 16. Available from: https://doi.org/10.1186/ 2008-2231-22-16. Xiang, M., Qian, Z.Y., Zhou, C.H., Liu, J., Li, W.N., 2006a. Crocetin inhibits leukocyte adherence to vascular endothelial cells induced by AGEs. J. Ethnopharmacol. 107, 25
31. Xiang, M., Yang, M., Zhou, C., Liu, J., Li, W., Qian, Z., 2006b. Crocetin prevents AGEs-induced vascular endothelial cell apoptosis. Pharm. Res. 54, 268
274. Xiang, M., Yang, R., Zhang, Y., Wu, P., Wang, L., Gao, Z., et al., 2017. Effect of crocetin on vascular smooth muscle cells migration induced by advanced glycosylation end products. Microvasc. Res. 112, 30
36. Xu, G., Qian, Z., Yu, S., Gong, Z., Shen, X., 2006. Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro. J. Asian Nat. Prod. Res. 79, 85
88. Zheng, S., Qian, Z., Tang, F., Sheng, L., 2005. Suppression of vascular cell adhesion molecule-1 expression by crocetin contributes to attenuation of atherosclerosis in hypercholesterolemic rabbits. Biochem. Pharmacol. 70, 1192
1199. Zheng, S., Qian, Z., Sheng, L., Wen, N., 2006. Crocetin attenuates atherosclerosis in hyperlipidemic rabbits through inhibition of LDL oxidation. J. Cardiovasc. Pharmacol. 47, 70
76. Zhou, C.H., Qian, Z.Y., Zheng, S.G., Xiang, M., 2006. ERK1/2 pathway is involved in the inhibitory effect of crocetin on angiotensin II-induced vascular smooth muscle cell proliferation. Eur. J. Pharmacol. 535, 61
68. Zhou, C.H., Qian, Z.Y., Xiang, M., He, S.Y., 2007. Involvement of Ca21 in the inhibition by crocetin of angiotensin II-induced ERK1/2 activation in vascular smooth muscle cells. Eur. J. Pharm. 554, 85
91. Zhou, C.H., Xiang, M., He, S.Y., Qian, Z.Y., 2010a. Crocetin inhibits cell cycle G1/S transition through suppressing cyclin D1 and elevating p27kip1 in vascular smooth muscle cells. Phytother. Res. 24, 975
981. Zhou, C.H., Xiang, M., He, S.Y., Qian, Z.Y., 2010b. Protein kinase C pathway is involved in the inhibition by crocetin of vascular smooth muscle cells proliferation. Phytother. Res. 24, 1680
1686. Zhou, T., Chuang, C., Zuo, L., 2015. Molecular characterization of reactive oxygen species inmyocardial ischemia-reperfusion injury. Biomed. Res. Int. 2015, 8649. Available from: https://doi.org/10.1155/2015/864946.

Chapter 30

Saffron, its main derivatives, and their effects on the respiratory system Mohammad-Hossein Boskabady1,2, Vahideh Ghorani1,2 and Azam Alavinezhad1,2 1

Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 2Department of Physiology, School of

Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 30.1 Introduction 30.2 Bronchodilatory effect of saffron and its derivatives 30.2.1 Relaxant effect of saffron extracts on airway smooth muscle 30.2.2 Relaxant effect of saffron derivatives on airway smooth muscle 30.2.3 Possible mechanisms of the airway smooth muscle relaxant effect of saffron and its derivatives

30.1

461 462 462 462

30.3 Prophylactic effect of saffron and its derivatives on respiratory disorders 30.3.1 Prophylactic effect of saffron extracts on respiratory disorders 30.3.2 Prophylactic effect of saffron derivatives on respiratory disorders 30.4 Conclusion References

464 464 466 467 467

463

Introduction

Saffron (Crocus sativus L.) has been regarded traditionally as a flavoring agent with very low toxicity. Although doses of less than 1.5 g of this plant are nontoxic for human, doses of more than 5 g may be toxic, and doses of at least 20 g day21 could be lethal (Kianbakht, 2008). In clinical trials, the commonly applied doses are lower than 30
50 mg day21 (Kianbakht, 2008; Moshiri et al., 2015). Pharmacokinetic studies of main derivatives of saffron such as crocin and crocetin have shown that crocin could not be absorbed via the gastrointestinal tract while crocetin can be absorbed after oral administration and has short plasma half-life (Asai et al., 2005; Xi et al., 2007). Saffron stigmata have long been used in traditional medicine. Biomedical research has demonstrated the many pharmacological effects of this plant, as well as its derivatives. Saffron is effective in the treatment of many disorders such as neurodegenerative diseases, memory impairment, depression (Abe and Saito, 2000; Noorbala et al., 2005; Sugiura et al., 1995), ischemic retinopathy (Xuan et al., 1999), and inflammatory diseases (Hosseinzadeh and Younesi, 2002). Saffron petal extract has various properties including antihypertensive (Fatehi et al., 2003), antioxidant (Assimopoulou et al., 2005; Hosseinzadeh et al., 2009), genoprotective and carcinogenesis prevention effects (Abdullaev, 2002). Several effects have been reported for saffron and its derivatives on respiratory tract, such as antitussive (Hosseinzadeh and Ghenaati, 2006), antimicrobial (Pintado et al., 2011), relaxant effect on trachea smooth muscle, stimulatory effect on β2-adrenoceptors, inhibitory effect on histamine H1 receptors of tracheal smooth muscle (Keyhanmanesh et al., 2010; Nemati et al., 2008), as well as antiinflammatory and immunomodulatory in experimental lung diseases

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00031-9 © 2020 Elsevier Inc. All rights reserved.

461

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(Boskabady and Farkhondeh, 2016). Therefore, in this chapter, the various pharmacological effects of saffron and its derivatives in the respiratory system will be presented.

30.2

Bronchodilatory effect of saffron and its derivatives

Several studies assessed the bronchodilatory activity of saffron and its derivatives and indicated that this property is attributed to the relaxant effect that it has on airway smooth muscles. The following are the possible mechanisms by which saffron can enact a relaxation effect on these muscles.

30.2.1 Relaxant effect of saffron extracts on airway smooth muscle The relaxant effects of four cumulative concentrations (0.15, 0.3, 0.45, and 0.60 g %) of the aqueous-ethanolic extract of saffron on guinea pig tracheal smooth muscle were examined. The results indicated a relatively potent relaxant effect on tracheal smooth muscles when compared to the effect of theophylline (Boskabady and Aslani, 2006; Mokhtari-Zaer et al., 2015). On the other hand, Byrami et al. (2013) demonstrated that hydroethanolic extract of saffron decreased tracheal responsiveness to methacholine in sensitized guinea pigs (Byrami et al., 2013), which could be an indicator of saffron’s relaxant effect on airway smooth muscle. The antitussive effect observed for ethanolic extract of saffron also is suggested to be due to its dilatory effect on the airways (Hosseinzadeh and Ghenaati, 2006). The relaxant effects of saffron extracts on other smooth muscles including vascular, gastrointestinal, and urogenital smooth muscles (Boskabady et al., 2008; Chang et al., 1964; Fatehi et al., 2003; Imenshahidi et al., 2010, 2013) could support the relaxant effect on airway smooth muscle. In a study of a rat-isolated vas deferens and a guinea pig ileum, the relaxant action of the saffron petal extract on contractions induced by electrical field stimulation was also observed in the reduced smooth muscle tonicity (Fatehi et al., 2003). Also, the relaxant effect of the saffron extract on the contraction of uterine was reported (Chang et al., 1964). The results of another study also illustrated a potent inhibitory effect of the aqueous-ethanol saffron extract on heart rate (HR) and contractility by demonstrating how calcium channels are blocked in a guinea pig-isolated heart (Boskabady et al., 2008). Fatehi et al. reported the effects of the aqueous and ethanol petals’ extracts on mean arterial blood pressure (MABP) in anesthetized rats. There was a significant decrease in MABP of the rats treated with ethanol (30 mg 100 g21) or aqueous extract of saffron (50 mg 100 g21) (Fatehi et al., 2003). Similarly, the effect of aquatic saffron stigma extract on MABP in deoxycorticosterone acetate (DOCA) salt-induced hypertensive rats was shown. It was observed that saffron extract decreased MABP in DOCA salt-treated rats (Imenshahidi et al., 2010, 2013). The observed effects of saffron extract in these studies seem to be related to relaxation of the vascular and heart smooth muscle cells. Table 30.1 summarizes the relaxant effects of saffron on tracheal smooth muscle.

30.2.2 Relaxant effect of saffron derivatives on airway smooth muscle It was observed that three cumulative concentrations (4, 8, and 16 mg mL21) of safranal (one of the derivatives of saffron) reduced airway responsiveness in sensitized guinea pigs. There was a significant decrease of tracheal responsiveness in animals treated with safranal compared to untreated guinea pigs, which may be due to the relaxant effect of this substance on airway smooth muscles (Boskabady et al., 2014). Additionally, the relaxant effect of safranal was examined by assessment of the various effects of its four different concentrations (0.15, 0.30, 0.45, and 0.60 mL of 0.2 mg mL21 solution) on guinea pig tracheal smooth muscle. The results of this study indicated a concentration-dependent relaxant effect of safranal on tracheal smooth muscle (Boskabady and Aslani, 2006). It was also suggested that the antitussive effect of safranal could be due, at least in part, to its airway dilatory effect (Hosseinzadeh and Ghenaati, 2006). In another study, the relaxant effects of three concentrations of crocin (30, 60, and 120 μM mL21) on tracheal smooth muscle were tested. The results indicated a relatively potent relaxant effect of crocin on tracheal smooth muscle, however, one that is less than the effect of theophylline at studied concentrations (Saadat et al., 2019). Several studies have also reported the relaxant effect of various derivatives of saffron on other smooth muscles, including vascular smooth muscles (Duarte et al., 1993; Imenshahidi et al., 2010, 2014; Razavi et al., 2013; Tang et al., 2006; Xu et al., 2006, 2007, 2015), which could support their relaxant effect on airway smooth muscle. Imenshahidi et al. (2010, 2014) indicated that chronic treatment of DOCA salt-induced hypertensive rats with three doses of crocin (50, 100, and 200 mg kg21 day21) and safranal (0.25, 0.5, and 1 mg kg21) led to reduced MABP in a dose-dependent manner. The results illustrated that crocin and safranal possess antihypertensive properties that

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TABLE 30.1 Tracheal smooth muscle relaxant effects of saffron and its derivatives. Ext./Cons.

Effect

Experimental design

References

HEE

Relaxant effect on TSM

Meth-TSM contraction

Boskabady and Aslani (2006)

KCL-TSM contraction Incubated with Chlo 1 Pro 1 Atr, KCL-TSM contraction, in guinea pig model Reduction effect on TR to methacholine

Tracheal chain of OVA-sensitized guinea pig

Byrami et al. (2013)

Stigma EE

Antitussive effect

i.p. injection in guinea pigs exposed to a nebulized aqueous solution of citric acid 20%

Hosseinzadeh and Ghenaati (2006)

Safranal

Relaxant effect on TSM

Meth-TSM contraction

Boskabady and Aslani (2006)

KCL-TSM contraction Incubated with Chlo 1 Pro 1 Atr, KCL-TSM contraction, in guinea pig model

Crocin

Antitussive effect

i.p. injection in guinea pigs exposed to a nebulized aqueous solution of citric acid 20%

Hosseinzadeh and Ghenaati (2006)

Reduction effect on TR to Meth and OVA

Tracheal chain of OVA-sensitized guinea pig

Boskabady et al. (2014)

Relaxant effect on TSM

Meth-TSM contraction

Saadat et al. (2019)

KCL-TSM contraction Incubated with Chlo 1 Pro 1 Atr 1 Indo 1 Diltiaz 1 Gliben, KCL-TSM contraction, in rat model Atr, Atropine; Chlo, chlorpheniramine; Cons, derivatives; Diltiaz, diltiazem; EE, ethanolic extract; Ext, extract; Gliben, glibenclamide; HEE, hydroethanolic extract; Indo, indomethacin; i.p., intraperitoneal; Meth, methacholine; OVA, ovalbumin; Pro, propranolol; TR, tracheal responsiveness; TSM, tracheal smooth muscle.

normalizes blood pressure; this effect of safranal was higher than that of crocin. In another study, crocin improved the toxicity effects of diazinon on systolic blood pressure (SBP) and HR in rats. The improvement of SBP and HR could be due to the relaxant effect of crocin on muscle cells (Razavi et al., 2013). The relaxation effects of other derivatives of saffron such as crocetin, quercetin, and kaempferol were also demonstrated. There is some evidence that crocetin (15, 30 mg kg21) improves endothelium-dependent relaxation in the thoracic aorta in hypercholesterolemic rabbit (Tang et al., 2006). Duarte et al. (1993) assessed the vasodilatory effect of quercetin and kaempferol on rat aortic smooth muscle. The results of this assessment demonstrated that these substances relaxed rat aortic strips that were contracted by noradrenaline, potassium chloride (KCl) or phorbol 12-myristate, 13acetate through the inhibition of protein kinase C (Duarte et al., 1993). In another study, the relaxation effects of quercetin and kaempferol were further demonstrated using porcine coronary arteries (Xu et al., 2007). The vascular effects of kaempferol (1 nM
100 μM) in isolated porcine coronary artery rings were examined, and the concentration-relaxation curve was recorded. Kaempferol enhanced endothelium-independent and -dependent relaxation at high concentrations. It was also shown that kaempferol enhanced endothelium-dependent relaxation in porcine coronary artery smooth muscle cells by activation of large-conductance Ca21-activated K1 channels (Xu et al., 2006, 2015).

30.2.3 Possible mechanisms of the airway smooth muscle relaxant effect of saffron and its derivatives Several studies focused on possible mechanisms responsible for the relaxant effect of saffron and its derivatives on various smooth muscles. The results of these studies indicated that the major mechanisms involved in tracheal smooth

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muscle, especially with safranal, are the stimulation of β2-adrenergic receptors, inhibition of the histamine H1 and muscarinic receptors, and potassium channel-opening effect. In a study conducted by Nemati et al. (2008), the stimulatory effect of the aqueous-ethanolic saffron and safranal extract was tested on β2-adrenoceptors in tracheal smooth muscle by performing concentration-response curves of isoprenaline on precontracted smooth muscle. The results demonstrated that the isoprenaline curves shift to the left in the presence of the extract, safranal, and propranolol. These findings indicate the stimulatory effects of saffron extract and safranal on β2-adrenoreceptors (Nemati et al., 2008). Additionally, the effect of saffron and safranal extract on muscarinic receptors of guinea pig tracheal smooth muscles was examined. The methacholine concentration-response curve shifts to the right in the presence of safranal extract and indicated a muscarinic receptor-blocking effect in this study (Neamati and Boskabady, 2010). Another responsible mechanism for the relaxant effect of saffron and safranal on airway smooth muscles is their inhibitory effect on histamine H1 receptors. To examine the histamine H1 receptors’ inhibitory effect, the effects of aqueous-ethanolic extract of saffron (0.025%, 0.05%, and 0.1%) and safranal (0.63, 1.25, and 2.5 μg mL21) on the histamine concentration-response curve was recorded in the presence of saline, saffron extract, safranal, and chlorpheniramine. The effects of saffron extract and safranal on the curve were similar to the effect of chlorpheniramine because they shifted the histamine-response curve to the right. Therefore, both the extract and its constituent showed the competitive antagonistic effect on histamine H1 receptors (Boskabady et al., 2010, 2011a). In another study investigating the possible mechanism of the relaxant effect of the saffron extract on tracheal smooth muscles, nonincubated tissues and tissues incubated with atropine, propranolol, and chlorpheniramine tissues were contracted by KCl. The relaxant effect of saffron extract was then examined. The relaxant effect of extract in the incubated tissues was less when compared to the nonincubated tissues, and demonstrated the possible inhibitory effect of the saffron extract on muscarinic and histamine H1 receptor as well as the possible stimulatory effect on β2-adrenoceptors (Boskabady and Aslani, 2006). The possible mechanisms of crocin’s relaxant effect on airway smooth muscles were also evaluated by a method similar to what was previously described. Nonincubated tissues and tissues incubated with atropine, chlorpheniramine, indomethacin, diltiazem, glibenclamide, and propranolol were contracted with 60 mM KCl, and the relaxant effect of crocin on these tissues was examined. The results indicated that the responsible mechanisms for the relaxant effect of crocin on tracheal smooth muscles likely include muscarinic receptor blocking, potassium channel opening, and ß2adrenoreceptors stimulation (Saadat et al., 2019). The possible mechanisms of tracheal smooth muscle relaxant effects of saffron and its derivatives are summarized in Table 30.2.

30.3

Prophylactic effect of saffron and its derivatives on respiratory disorders

The prophylactic effect of saffron and its derivatives has been reported in studies of various animals with respiratory disorders.

30.3.1 Prophylactic effect of saffron extracts on respiratory disorders The prophylactic effect of the saffron extract on inflammatory changes in ovalbumin (OVA)- sensitized guinea pigs was evaluated. The preventive effect of various concentrations of saffron extract (0.1, 0.2, and 0.4 mg mL21) on total and differential count of white blood cells (WBCs) in the blood of sensitized guinea pigs (Bayrami and Boskabady, 2012; Boskabady and Farkhondeh, 2016) was shown. In a similar study, the preventive effect of saffron extract and safranal on lung pathology, total and differential WBC count in lung lavage, and the serum level of histamine in sensitized guinea pigs were examined. The extract improved pathological changes, total and differential WBC count, and serum histamine level. However, the preventive effect of safranal was more prominent than the effect of the extract (Boskabady et al., 2012). Another study also confirmed similar effects of hydroalcoholic extract of saffron on bronchial inflammatory cells in OVA-sensitized rats. In this study, pretreatment of sensitized animals with 50, 100, and 200 mg kg21 of saffron extract led to reduction in the total and differential WBC in lavage when compared to that of untreated rats (Mahmoudabady et al., 2013). The prophylactic effect of the saffron extract was also examined on tracheal responsiveness, cytokines, and total NO and nitrite in sensitized guinea pigs. The preventive effect of saffron at various concentrations was indicated by the increased IFN-γ serum level and IFN-γ/IL-4 ratio and by a reduction of tracheal responsiveness, serum level of IL-4, and total NO and nitrite after treatment of sensitized guinea pigs (Byrami et al., 2013). The prophylactic effect of

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TABLE 30.2 Possible mechanisms of tracheal smooth muscle relaxant effects of saffron and its derivatives. Ext./Cons.

Effect

Experimental design

References

HEE

Stimulatory effect on β2adrenoceptors

CLCRC of Isop. of guinea pig TC, in the presence of HEE and Prop.

Nemati et al. (2008)

Inhibitory effect on muscarinic receptor

CLCRC of Meth. of guinea pig TC, in the presence of HEE and Atr.

Neamati and Boskabady (2010)

Inhibitory effect on histamine (H1) receptor

CLCRC of Hist. of guinea pig TC, in the presence of HEE and Chlo.

Boskabady et al. (2010)

Stimulatory effect on β2adrenoceptors

KCl-and Meth.-induced contraction of TSM in guinea pig model

Boskabady and Aslani (2006)

Stimulatory effect on β2adrenoceptors

CLCRC of Isop. of guinea pig TC, in the presence of safranal and Prop.

Nemati et al. (2008)

Inhibitory effect on muscarinic receptor

CLCRC of Meth. of guinea pig TC, in the presence of safranal and Atr.

Neamati and Boskabady (2010)

Inhibitory effect on histamine (H1) receptor

CLCRC of Meth. of guinea pig TC, in the presence of HEE and Chlo.

Boskabady et al. (2011a)

Stimulatory effect on β2adrenoceptors

KCl-and Meth.-induced contraction of TSM in guinea pig model

Boskabady and Aslani (2006)

KCl-and Meth.-induced contraction of TSM in a rat model

Saadat et al. (2019)

Inhibitory effect on muscarinic receptor Inhibitory effect on histamine (H1) receptor Safranal

Inhibitory effect on muscarinic receptor Inhibitory effect on histamine (H1) receptor Crocin

Stimulatory effect on β2adrenoceptors Inhibitory effect on muscarinic receptor Potassium channel-opening effect

Atr, Atropine; Chlo, chlorpheniramine; CLCRC, cumulative log concentration-response curve; Cons, derivatives; Ext, extract; HEE, hydroethanolic extract; Isop, isoprenaline; Meth, methacholine; Pro, propranolol; TC, tracheal chain; TSM, tracheal smooth muscle.

hydroalcoholic saffron extract on asthmatic rats also showed reduced WBC, red blood cell, and platelet count (Vosooghi et al., 2013). Furthermore, the preventive effect of the saffron extract on inflammatory markers such as serum levels of endothelin-1 (ET-1) and total protein (TP) in sensitized guinea pigs was studied. Findings indicated that serum levels of ET-1 and TP were significantly reduced in animals pretreated with the extract when compared to untreated guinea pigs (Gholamnezhad et al., 2013). The effect of saffron extract (50, 250, and 500 g mL21) on cell viability and various cytokines in nonstimulated and stimulated human lymphocytes was also examined. Improvement of inflammatory conditions in increased IFN-γ/IL-4 ratio was demonstrated, which indicates an increased Th1/Th2 balance (Boskabady et al., 2011b), showing the preventive effect of saffron on inflammatory process and immune dysregulation in sensitized animals. In another study, ethanolic extract of saffron stigma (100
800 mg kg21) was injected intraperitoneally in guinea pigs to assess the antitussive effect of the extract. The results indicated that ethanolic extract significantly reduced cough numbers in guinea pigs while aqueous extract of saffron did not show such effect (Hosseinzadeh and Ghenaati, 2006). In Table 30.3, the prophylactic effects of saffron on respiratory disorders are summarized.

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TABLE 30.3 Prophylactic effects of saffron and its derivatives on respiratory disorders. Ext./Cons.

Effect

Experimental design

References

HEE

Preventive effect on blood total and differential WBC counts

OVA-sensitized guinea pigs

Bayrami and Boskabady (2012)

Improved pathological changes, lung lavage total, and differential WBC and histamine serum level

OVA-sensitized guinea pigs

Boskabady et al. (2012)

Reduced total and differential WBC in lung lavage

OVA-sensitized rats

Mahmoudabady et al. (2013)

Preventive effect on tracheal responsiveness and serum levels of inflammatory mediators and increased Th1/Th2 balance

OVA-sensitized guinea pigs

Byrami et al. (2013)

Reduced WBC, RBC and platelet count

OVA-sensitized rats

Vosooghi et al. (2013)

Ameliorated ET-1 and TP levels

OVA-sensitized guinea pigs

Gholamnezhad et al. (2013)

Increased IFN-γ/IL-4 ratio or Th1/Th2 balance

Nonstimulated and PHAstimulated human lymphocytes

Boskabady et al. (2011b)

Stigma EE

Reduced cough numbers

i.p. injection in guinea pigs exposed to a nebulized aqueous solution of citric acid 20%

Hosseinzadeh and Ghenaati (2006)

Safranal

Preventive effect on blood total and differential WBC counts

OVA-sensitized guinea pigs

Bayrami and Boskabady (2012)

Improved pathological changes, lung lavage total, and differential WBC and histamine serum level

OVA-sensitized guinea pigs

Boskabady et al. (2012)

Ameliorated ET-1 and TP levels

OVA-sensitized guinea pigs

Gholamnezhad et al. (2013)

Reduced cough numbers

i.p. injection in guinea pigs exposed to a nebulized aqueous solution of citric acid 20%

Hosseinzadeh and Ghenaati (2006)

Prophylactic effect as elevated Th1/Th2 balance and reduced tracheal responsiveness, serum levels of IL-4 and total NO and nitrite

OVA-sensitized guinea pigs

Boskabady et al. (2014)

Increased IFN-γ/IL-4 ratio or Th1/Th2 balance

Nonstimulated and PHAstimulated human lymphocytes

Feyzi et al. (2016)

Reduced WBC, inflammatory mediators and MAPK in lung

OVA-sensitized mice

Xiong et al. (2015)

Crocin

Cons, Derivatives; EE, ethanolic extract; ET-1, endothelin-1; Ext, extract; HEE, hydroethanolic extract; IFN-γ, interferon-γ; IL, interleukin; i.p., intraperitoneal; MAPK, MAP kinase; OVA, ovalbumin; PHA, phytohemagglutinin; RBC, red blood cell; Th, T helper; TP, total protein; WBC, white blood cell.

30.3.2 Prophylactic effect of saffron derivatives on respiratory disorders The effect of safranal on guinea pigs sensitized with OVA showed a preventive effect of the different concentrations of safranal on total and differential count of WBC in the blood (Bayrami and Boskabady, 2012; Boskabady and Farkhondeh, 2016). Similarly, the effect of safranal produced improvement in lung pathological changes, total and differential WBC count in lung lavage, and the serum level of histamine in sensitized guinea pigs (Boskabady et al.,

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467

2012). The prophylactic effect of various concentrations of safranal on tracheal responsiveness, serum levels of cytokines, and total NO and nitrite in sensitized guinea pigs indicated increased IFN-γ level and IFN-γ/IL-4 ratio but a reduction of tracheal responsiveness, level of serum IL-4, and total NO and nitrite in sensitized guinea pigs (Boskabady et al., 2014). Safranal also showed significant effect on inflammatory markers such as serum levels of ET-1 and TP in the sensitized guinea pig by reducing these markers (Gholamnezhad et al., 2013). The effect of safranal (0.1, 0.5, and 1 mM) on various cytokines in nonstimulated and stimulated human lymphocytes showed increased IFN-γ/IL-4 ratio, indicating an increased Th1/Th2 balance (Feyzi et al., 2016). Further experiments performed on safranal illustrated an antitussive effect for safranal (0.25
0.75 mL kg21) in guinea pigs exposed to a nebulized aqueous solution of 20% citric acid, while crocin (another constituent of saffron) did not show this effect (Hosseinzadeh and Ghenaati, 2006). The preventive effect of crocin as an antiasthma agent in a murine model was studied. In this study, OVA-sensitized and -challenged mice were administrated crocin one hour before every OVA challenge. Total and differential WBC of lavage, inflammatory mediators in serum, and BALF in lung as well as MAPK signaling pathway were evaluated. The results indicated that crocin reduced numbers of WBC count, levels of inflammatory mediators, and phosphorylated MAP kinases in lung tissues (Xiong et al., 2015).

30.4

Conclusion

This chapter summarized several studies regarding the relaxant (bronchodilatory) and prophylactic effects of saffron and some of its derivatives on the respiratory system. Based on the aforementioned studies, it is concluded that saffron, safranal, and crocin showed the relaxant effects on smooth trachea muscles, which indicate their possible bronchodilatory effects on obstructive pulmonary diseases. The possible mechanisms of the relaxant effect of saffron, safranal, and crocin on airway smooth muscle include the stimulation of β2-adrenergic receptors, inhibition of histamine H1 and muscarinic receptors, and potassium channel-opening. The prophylactic effect of saffron, safranal, and crocin on respiratory disorders (namely, asthma) was described, and the antitussive effects of the respective extracts were documented. Conclusively, saffron and its derivatives could be possible candidates for the treatment of obstructive pulmonary diseases for both relieving (bronchodilatory effect) therapy and preventive (prophylactic effect) therapy.

References Abdullaev, F.I., 2002. Cancer chemopreventive and tumoricidal properties of saffron (Crocus sativus L.). Exp. Biol. Med. 227, 20
25. Abe, K., Saito, H., 2000. Effects of saffron extract and its constituent crocin on learning behavior and long-term potentiation. Phytother. Res. 14, 149
152. Asai, A., Nakano, T., Takahashi, M., Nagao, A., 2005. Orally administered crocetin and crocins are absorbed into blood plasma as crocetin and its glucuronide conjugates in mice. J. Agric. Food Chem. 53, 7302
7306. Assimopoulou, A., Sinakos, Z., Papageorgiou, V., 2005. Radical scavenging activity of Crocus sativus L. extract and its bioactive derivatives. Phytother. Res. 19, 997
1000. Bayrami, G., Boskabady, M.H., 2012. The potential effect of the extract of Crocus sativus and safranal on the total and differential white blood cells of ovalbumin-sensitized guinea pigs. Res. Pharm. Sci. 7 (4), 249
255. Boskabady, M.H., Aslani, M.R., 2006. Relaxant effect of Crocus sativus (saffron) on guinea-pig tracheal chains and its possible mechanisms. J. Pharm. Pharmacol 58, 1385
1390. Boskabady, M.H., Farkhondeh, T., 2016. Antiinflammatory, antioxidant, and immunomodulatory effects of Crocus sativus l. and its main derivatives. Phytother. Res. 30, 1072
1094. Boskabady, M.H., Shafei, M., Shakiba, A., Sefidi, H.S., 2008. Effect of aqueous-ethanol extract from Crocus sativus (saffron) on guinea-pig isolated heart. Phytother. Res. 22, 330
334. Boskabady, M.H., Rahbardar, M.G., Nemati, H., Esmaeilzadeh, M., 2010. Inhibitory effect of Crocus sativus (saffron) on histamine (H1) receptors of guinea pig tracheal chains. Pharmazie. 65, 300
305. Boskabady, M.H., Rahbardar, M.G., Jafari, Z., 2011a. The effect of safranal on histamine (H1) receptors of guinea pig tracheal chains. Fitoterapia 82, 162
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1545. Boskabady, M.H., Tabatabaee, A., Byrami, G., 2012. The effect of the extract of Crocus sativus and its constituent safranal, on lung pathology and lung inflammation of ovalbumin sensitized guinea-pigs. Phytomedicine 19, 904
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535. Chang, P., Wang, C., Liang, J., Kuo, W., 1964. Studies on the pharmacological action of Zang Hong Hua (Crocus sativus L.). I. Effects on uterus and estrus cycle. Yao xue xue bao. Acta. Pharm. Sin. 11, 94
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862. Fatehi, M., Rashidabady, T., Fatehi-Hassanabad, Z., 2003. Effects of Crocus sativus petals’ extract on rat blood pressure and on responses induced by electrical field stimulation in the rat isolated vas deferens and guinea-pig ileum. J. Ethnopharmacol. 84, 199
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642. Razavi, M., Hosseinzadeh, H., Abnous, K., Motamedshariaty, V.S., Imenshahidi, M., 2013. Crocin restores hypotensive effect of subchronic administration of diazinon in rats. Iran J. Basic Med. Sci. 16 (1), 64
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152.

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Chapter 31

Saffron’s role in metabolic disorders Ahmad Ghorbani Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 31.1 Introduction 31.2 Effects of saffron on obesity 31.3 Effects of saffron on diabetes 31.3.1 Antihyperglycemic effects of saffron

31.1

471 471 472 473

31.3.2 Antihyperglycemic mechanisms of saffron 31.3.3 Effects of saffron on diabetic complications 31.4 Conclusion References

473 476 479 480

Introduction

Metabolic disorders are among the most common contemporary diseases in the human population (Golden et al., 2009). The global prevalence of these disorders, which include obesity and diabetes, has increased to approximately half a billion occurrences. The World Health Organization (WHO) reported that over 1.9 billion adults were overweight in 2014 and, of these, more than 600 million were obese. In that year, the global prevalence of diabetic patients was 422 million occurrences. Metabolic disorders, specifically hyperlipidemia, obesity, and diabetes, are often principal causative factors in the development of terminal conditions such as stroke and cardiovascular disease (Nelson, 2013; Peters et al., 2014; Shah et al., 2015). For thousands of years and in many countries, medicinal plants have been used to treat various human diseases. Pharmacological studies have confirmed the effectiveness of a number of medicinal plants in managing metabolic and endocrine disorders such as metabolic syndrome, diabetes, obesity, and thyroid dysfunction (Dos Santos et al., 2011; Ghorbani, 2013a,b, 2014; Hasani-Ranjbar et al., 2013; Kar et al., 2002). In this chapter, data from experimental and clinical studies evaluating the beneficial effects of saffron (Crocus sativus L.) on obesity and diabetes have been summarized. Additionally, the putative mechanisms by which saffron induces these beneficial effects are discussed.

31.2

Effects of saffron on obesity

Being overweight or obese is defined as having excessive fat accumulation in the body to the extent that health is adversely affected. According to WHO’s classification, subjects with a body mass index (BMI) measurement greater than or equal to 25 and 30 are considered overweight or obese, respectively (Seidell, 2005). Obesity is among the most common metabolic disorders worldwide. In addition to lifestyle and diet, pharmacotherapy interventions can be considered for the treatment of obesity. Overall, the pharmacological treatment choices for obesity are, at present, limited. The currently available drugs can be categorized into three groups: those that decrease food intake (e.g., phentermine and sibutramine); those that inhibit intestinal fat absorption (e.g., orlistat); and those that induce thermogenesis (e.g., ephedrine and caffeine). Monoamines acting on noradrenergic receptors, serotonin receptors, dopamine receptors, and histamine receptors can reduce food intake (Bray, 2000). However, the overall efficacy of these drugs in long-term weight maintenance is somewhat limited. Also, the long-term safety of these drugs still remains to be clarified by additional studies. Currently, diet-based therapies and medicinal plant supplements are among the most commonly paired alternative types of treatment for weight loss (Hasani-Ranjbar et al., 2013). Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00032-0 © 2020 Elsevier Inc. All rights reserved.

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There are several reports showing that saffron derivatives have beneficial effects on obesity (Gout et al., 2010; Hazman et al., 2016; Kianbakht and Hashem-Dabaghian, 2015; Mashmoul et al., 2014, 2017; Sheng et al., 2006). In an experimental study on rats that received a high-fat diet, daily administration of crocin (150 mg kg21, oral) for 6 weeks decreased weight gain (Hazman et al., 2016). In another study, Kianbakht and Hashem-Dabaghian (2015) observed that crocin (5
50 mg kg21) and saffron alcoholic extract (25
200 mg kg21) reduced food intake and body weight. These effects of the crocin and saffron extract were comparable to the effects of sibutramine. Similarly, Mashmoul et al. (2014) showed that alcoholic extract of saffron (80 mg kg21) significantly decreased food consumption and crocin (80 mg kg21) reduced the rate of weight gain in rats fed a high-fat diet. Crocin was also able to decrease the ratio of fat mass to body weight in obese rats as well as the serum level of triglyceride (TG). Saffron extract decreased the level of total cholesterol (TC) and the ratio of low-density lipoprotein (LDL) to high-density lipoprotein (HDL) as the atherogenic index (Mashmoul et al., 2014). In this study, orlistat was more effective compared to crocin in terms of reducing weight gain and fat absorption. Previous studies have shown that orlistat inhibits the activities of both gastric and pancreatic lipases, while crocin has a higher selectivity for pancreatic lipase. Also, the inhibitory effect of orlistat on lipase is irreversible, however the inhibition of crocin is reversible (Sheng et al., 2006). A mild but statistically significant decrease in weight gain was also observed with the extract of saffron stigma in a randomized placebo-controlled study on mildly overweight (but otherwise healthy) subjects (Gout et al., 2010). It has been reported that saffron extract has no significant effect on serum lipids of patients with type-2 diabetes (T2D) (Milajerdi et al., 2018), so the effects of saffron or its phytochemicals on serum lipids may be different between nondiabetic and diabetic subjects. Two hormones, ghrelin and leptin, play important roles in the regulation of energy homeostasis in the body. Ghrelin, a stomach-derived polypeptide, enhances appetite and therefore regulates food intake, body weight, and adiposity (Mu¨ller et al., 2015). Leptin is a polypeptide hormone that is produced by adipocytes and circulates at concentrations proportional to body fat mass. Leptin reduces food intake and body weight; obese subjects have elevated plasma leptin levels, raising the possibility that obesity is associated with leptin resistance (Myers et al., 2010; Schwartz et al., 2017). Therefore, hyperleptinemia may be considered as an index of excessive body fat mass (Shah and Braverman, 2012). There are reports that saffron and crocin could decrease the blood leptin level in obese and T2D animals (Kianbakht and Hashem-Dabaghian, 2015; Mashmoul et al., 2017), however, some studies observed no significant effect of saffron and crocin on the leptin level (Bajerska et al., 2013; Hazman et al., 2016). Therefore, the exact effect of saffron on the leptin remained to be elucidated. Crocin was shown to significantly increase the plasma level of gherlin, which functions in the brain to prompt adiposity (Mashmoul et al., 2017). In many tissues, obesity is associated with the activation of inflammatory processes and an increased level of proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) (Schwartz et al., 2017). Oxidative stress is considered as one of the potential inducers of inflammatory processes as well as vulnerability to obesity and related disorders (Ferna´ndez-Sa´nchez et al., 2011; Zulet et al., 2007). The antiinflammatory and antioxidant properties of saffron have been well documented by several studies (Mashmoul et al., 2013; Poma et al., 2012). The findings of the study conducted by Hazman et al. (2016) demonstrated that crocin could be effective in reducing plasma level IL1β. Also, Mashmoul et al. (2017) found that plasma TNF-α level decreased after an 8-week administration of saffron extract. On the other hand, saffron extract and crocin significantly increased the plasma level of antiinflammatory mediator adiponectin. In summary, saffron may directly or indirectly inhibit obesity pathophysiology by decreasing food intake, weight gain, inflammatory possesses, oxidative stress, and reversing alterations in the levels of adipocytokines.

31.3

Effects of saffron on diabetes

Diabetes mellitus is one of the most common illnesses of the endocrine system. It is generally categorized into two main types: type-1 diabetes (T1D), also known as insulin-dependent diabetes, and T2D, also known as noninsulindependent diabetes. While patients with T1D suffer from a state of insulin deficiency due to the destruction of pancreatic beta cells, patients with T2D show a state of insulin resistance and beta-cell dysfunction (Gan et al., 2012; Kahn, 2003). Eventually, both T1D and T2D result in serious complications involving different organs, including the kidney, heart, retina, and nervous system (Fowler, 2008). Presently, insulin and oral hypoglycemic drugs such as biguanides, thiazolidinediones, sulfonylureas, meglitinides, α-glucosidase inhibitors, and incretin-agonists are prescribed for managing diabetes (Lorenzati et al., 2010). Nevertheless, it remains difficult to achieve long-term euglycemia in diabetic patients, and most patients experience a number of complications over time (Gregg et al., 2014; Palmer et al., 2004; Tzoulaki et al., 2009). In addition, consumption of the available antidiabetic drugs is associated with several side effects including abdominal discomfort, acidosis, and hypoglycemia (Lorenzati et al., 2010). Investigations have therefore

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continued to develop new antidiabetic medications with lesser side effects and more effectiveness. An increasing line of evidence indicates that some functional foods and medicinal plants have beneficial effects on diabetes (Ghorbani, 2013a,b, 2014; Hosseini et al., 2015). Saffron has been an important subject of diabetes research because of its favorable biological activities, including its antioxidant, antiinflammatory, and antiatherosclerotic properties (Srivastava et al., 2010).

31.3.1 Antihyperglycemic effects of saffron The results of the studies examining the metabolic effects of saffron in diabetic animals are shown in Table 31.1. Different parts of the saffron plant, including the stigma and petals, reduce the level of fasting blood glucose (FBG). This effect was shown to be produced in a wide range of doses (from 25 to 600 mg kg21) in different treatment periods (between 2 and 20 weeks) (Elgazar et al., 2013; Hemmati et al., 2015b; Kianbakht and Hajiaghaee, 2011; Shirali et al., 2012). Also, the effect on FGB was shown to be produced by different extracts prepared from saffron stigma, including aqueous, alcoholic, and hydroalcoholic extracts (Hemmati et al., 2015b; Rahbani et al., 2011; Samarghandian et al., 2014b). The benefits of saffron extracts on the FBG level is accompanied by a decrease in the levels of HbA1c, weight loss, and serum-advanced glycation end product (AGE) (Dehghan et al., 2016; Elgazar et al., 2013; Kianbakht and Hajiaghaee, 2011; Samarghandian et al., 2014b; Shirali et al., 2012). There are contradictory reports on the effects of saffron on FBG level in nondiabetic animals. While two studies reported that saffron doses of 40 and 50 mg kg21 reduced FBG levels, two other studies showed that doses as large as 100 mg kg21 had no significant hypoglycemic effect (Ali et al., 2016; Arasteh et al., 2011; Dehghan et al., 2016; Rahbani et al., 2011). It has been reported that an intraperitoneal injection of saffron produced a greater reduction in blood glucose when compared to an oral administration (Mohajeri et al., 2008). In a randomized controlled clinical trial in patients with T2D, consumption of 3 glasses of black tea containing 1 g saffron stigma per day had no significant effect on FBG and HbA1c (Azimi et al., 2014). On the other hand, in another clinical trial with patients who have T2D, administration of saffron hydroalcoholic extract (15 mg, twice daily, for 8 weeks) significantly decreased the FBG level (Milajerdi et al., 2018). Therefore, it seems that the method of preparation of saffron extract, the dose level, and the method of administration are important determinants of its effectiveness to reduce the blood glucose level. The effects of different saffron preparations on measures of glycemic control warrant further clinical trial investigations. The antihyperglycemic effect of saffron can be attributed to its pharmacologically active derivatives, crocin and safranal (Ahmadi et al., 2017; Altinoz et al., 2014a; Asri-Rezaei et al., 2015; Farshid et al., 2016; Farshid and Tamaddonfard, 2015; Hazman and Bozkurt, 2015; Hazman and Ovalı, 2015; Jin et al., 2009; Kianbakht and Hajiaghaee, 2011; Maeda et al., 2014; Rajaei et al., 2013; Samarghandian et al., 2013, 2016; Shirali et al., 2013; Tamaddonfard et al., 2013). Crocin is a carotenoid compound found in the flowers of saffron that is primarily responsible for the color of saffron. Safranal is a monoterpene aldehyde that is responsible for the saffron aroma. Crocin and safranal show antihyperglycemic effects in both T1D and T2D animal models (Table 31.1). In the case of crocin, this effect was shown to be produced in a range of doses from 10 to 200 mg kg21, while safranal induced such effect at lower doses, namely 0.25 to 20 mg kg21 (Farshid et al., 2016; Jin et al., 2009; Kianbakht and Hajiaghaee, 2011; Maeda et al., 2014). In addition to the decreasing the FBG, both crocin and safranal have been shown to improve glucose tolerance, decrease HbA1c level, and inhibit weight loss (Ahmadi et al., 2017; Altinoz et al., 2014a; Asri-Rezaei et al., 2015; Farshid et al., 2016; Farshid and Tamaddonfard, 2015; Hazman and Bozkurt, 2015; Hazman and Ovalı, 2015; Jin et al., 2009; Kianbakht and Hajiaghaee, 2011; Maeda et al., 2014; Rajaei et al., 2013; Samarghandian et al., 2013, 2016; Shirali et al., 2013; Tamaddonfard et al., 2013). It was reported that crocin enhanced zinc’s ability to improve the serum levels of glucose and insulin in a streptozotocin (STZ) model of T1D (Asri-Rezaei et al., 2015). Additionally, crocin was shown to improve insulin resistance in the T2D animal model (Shirali et al., 2013). Studies have shown that safranal is more effective than glibenclamide, a sulfonylurea oral hypoglycemic drug, in reducing the levels of FBG and HbA1c (Kianbakht and Hajiaghaee, 2011). However, Farshid and Tamaddonfard (2015) reported that insulin’s blood glucose-lowering property was more prominent than those of crocin and safranal, and that crocin and safranal enhance insulin’s hypoglycemic effect in a rat with T1D. No difference was observed between crocin (30 mg kg21) and safranal (1 mg kg21) with respect to reducing FBG level.

31.3.2 Antihyperglycemic mechanisms of saffron The antihyperglycemic effect of phytochemicals can be achieved by different mechanisms which include decreasing the absorption of carbohydrates in the small intestine, enhancing insulin release from the pancreas, preventing tissue

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TABLE 31.1 Summary of experimental studies evaluated the metabolic effects of different parts of saffron in diabetes. Plant material

Study model

Treatment dosea and duration

Effects

References

Stigma

STZ model of T1D rats

40 mg kg21 (i.p.) of ethanolic extract for 8 weeks

kFBG; kAST; kALT; kALP; kBilirubin; kOxidative stress in the liver; kHistological damage in the liver

Rahbani et al. (2011, 2012)

STZ model of T1D rats

20, 40, and 80 mg kg21 (i.p.) of aqueous extract for 4 weeks

kFBG; kWeight lost; kTG; kTC; kLDL; mHDL; kSerum AGE; kSerum TNF-α; kOxidative stress in the hippocampus; kCognitive deficit

Samarghandian et al. (2014b)

STZ model of T1D rats

25 and 100 mg kg21 of hydroalcoholic extract for 2 weeks

kFBG;  TC; kTG; kVLDL; mSerum adiponectin

Hemmati et al. (2015b)

STZ model of T1D rats

100 mg kg21 (oral) of aqueous extract for 6 weeks

kFBG; mSerum insulin; kTotal lipids; kTC; kTG; kLDL; kVLDL; kAST; kALT; kTotal lipids, oxidative stress, AST and ALT in the liver

Ali et al. (2016)

STZ model of T1D rats

40 mg kg21 (oral) of hydroalcoholic extract for 6 weeks

kFBG; kHbA1c; kWeight lost; kInsulin resistance; kTC; kTG; kLDL; kVLDL;  HDL;  Leptin;  Adiponectin

Dehghan et al. (2016)

Alloxan model of T1D rats

20, 40, and 80 mg kg21 (i.p.) of ethanolic extract for 2 weeks

kFBG; mSerum insulin; kHistological damage in the pancreas

Mohajeri et al. (2008)

Alloxan model of T1D rats

80 and 240 mg kg21 (oral) of methanolic extract for 6 weeks

kFBG; kHbA1c; mSerum insulin;  ALT;  AST;  Creatinine

Kianbakht and Hajiaghaee (2011)

Alloxan model of T1D rats

200, 400, and 600 mg kg21 (oral) of aqueous extract for 4 weeks

kFBG; mSerum insulin; kWeight lost; kTG; kTC; kAST; kALT; kALP; kUrea; kCreatinine; kHistological damage in the pancreas

Elgazar et al. (2013)

HFD 1 STZ model of T2D rats

HFD containing 0.08% of the extract for 35 days

kFBG; mBeta cell function; mPancreas mass; kTG;  TC;  Leptin

Bajerska et al. (2013)

Fructose 1 STZ model of T2D rats

100 mg kg21 of total saffron for 2 months

kFBG; kAST; kALT; kALP; kTG;  TC;  Adiponectin; kHistological damage in the liver

Konstantopoulos et al. (2017)

STZ model of T2D rats

100 and 150 mg kg21 of aqueous extract for 5 months

kFBG; kHbA1c; kMortality; kWeight lost; kTG; kTC; kLDL; mHDL

Shirali et al. (2012)

Petals

STZ model of T1D rats

25 and 100 mg kg21 (oral) of aqueous and ethanolic extracts for 3 weeks

kFBG; kTG; kVLDL;  HDL; mSerum adiponectin

Hemmati et al. (2015a)

Crocin

STZ model of T1D rats

15, 30, and 60 mg kg21 (i.p.) for 6 weeks

kFBG; kOxidative stress in the liver, kidney and brain cortex; kLearning and memory impairment

Ahmadi et al. (2017), Rajaei et al. (2013)

STZ model of T1D rats

10, 20, and 30 mg kg21 per day (i.p.) for 4 weeks

kFBG; kWeight lost; kTG; kTC; kLDL; mHDL; kSerum oxidative stress markers; kInflammatory cytokine in the aorta

Samarghandian et al. (2016)

STZ model of T1D rats

12.5, 25, and 50 mg kg21 (i.p.) for 6 weeks

kFBG; mSerum insulin; kSerum oxidative stress markers

Asri-Rezaei et al. (2015) (Continued )

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TABLE 31.1 (Continued) Plant material

Safranal

Study model

Treatment dosea and duration

Effects

References

STZ model of T1D rats

30 mg kg21 (i.p.) for 56 days

kFBG; kNeuropathic pain; kSciatic nerve lesion severity; kOxidative stress in sciatic nerve

Farshid and Tamaddonfard (2015)

STZ model of T1D rats

7.5, 15, and 30 mg kg21 (i.p.) for 4 weeks

kFBG; mSerum insulin; kSerum oxidative stress markers; kHippocampal neurons number loss; kLearning and memory impairment

Tamaddonfard et al. (2013)

STZ model of T1D rats

20 mg kg21 (oral) for 3 weeks

kFBG; kTG; kTC; kVLDL; kOxidative stress in the brain and heart; kHistopathological damage in the heart; kHistopathological damage in cerebrum and cerebellum

Altinoz et al. (2014a, 2015)

STZ model of T1D rats

5, 10 and 20 mg kg21 (i. p.) 5 days a week for 8 weeks

kFBG; kWeight lost; kOxidative stress in the heart; kHistological damage in the heart

Farshid et al. (2016)

Alloxan model of T1D rats

50 and 150 mg kg21 (oral) for 6 weeks

kFBG; kHbA1c; mSerum insulin

Kianbakht and Hajiaghaee (2011)

STZ model of T2D rats

50 and 100 mg kg21 (i. p.) of aqueous extract for 5 months

kFBG; kHbA1c; kAGE; kMicroalbuminuria; kMortality; kWeight lost; kTG; kTC; kLDL; mHDL

Shirali et al. (2013)

HFD 1 STZ model of T2D rats

50, 100, and 200 mg kg21 (oral) for 4 weeks

kFBG; mGlucose tolerance; kTG; kTC; kLDL; mHDL

Jin et al. (2009)

Alloxan model of T1D rats

0.25 and 0.5 mg kg21 (oral) for 6 weeks

kFBG; kHbA1c; mSerum insulin

Kianbakht and Hajiaghaee (2011)

STZ model of T1D rats

1 mg kg21 (i.p.) for 56 days

kFBG; kNeuropathic pain; kSciatic nerve lesion severity; kOxidative stress in sciatic nerve tissue

Farshid and Tamaddonfard (2015)

STZ model of T1D rats

0.25, 0.5, and 0.75 mg kg21 (i.p.) for 4 weeks

kFBG; kWeight lost; kTG; kTC; kSerum oxidative stress markers

Samarghandian et al. (2013)

HFD 1 STZ model of T2D rats

b

0.2 mL kg21 per day for 4 weeks

kFBG; kSerum inflammatory and oxidative stress markers kInflammation and oxidative stress in the pancreas and kidney

Hazman and Bozkurt (2015), Hazman and Ovalı (2015)

KK-Ay/Kwl model of T2D mice

20 mg kg21 (oral) for 2 weeks

mGlucose tolerance

Maeda et al. (2014)

AGE, Advanced glycation end-product; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; FBG, fasting blood glucose; HDL, high-density lipoprotein; HFD, high-fat diet; i.p., intraperitoneal injection; LDL, low-density lipoprotein; STZ, streptozotocin; T1D, type-1 diabetes; T2D, type-2 diabetes; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein; m, increase; k, decrease;  , no significant effect. a Effective doses are bolded. b Dose of safranal was not mentioned in the paper.

gluconeogenesis, increasing glucose uptake by tissues, and protecting and regenerating pancreatic beta cells (Ghorbani, 2017; Hosseini et al., 2015; Hui et al., 2009; Shafiee-Nick et al., 2011; Tashakori-Sabzevar et al., 2016; Vahid et al., 2017). The putative mechanisms by which saffron compounds decrease blood glucose are shown in Fig. 31.1. Saffron and its active compounds have been shown to stimulate the secretion of insulin from beta cells and thus increase serum insulin (Ali et al., 2016; Arasteh et al., 2011; Bajerska et al., 2013; Dehghan et al., 2016; Kianbakht and Hajiaghaee, 2011; Mohajeri et al., 2008; Tamaddonfard et al., 2013). Studies showed that an aqueous extract of saffron increased the serum level of insulin in alloxan-induced diabetic rats (Elgazar et al., 2013). Administration of 40 mg kg21 of

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FIGURE 31.1 Schematic overview of the mechanisms by which saffron compounds may decrease blood glucose. Saffron compounds reduce hydrolysis of carbohydrates in the small intestine, stimulate insulin release from pancreatic beta cells, protect beta cells against degeneration, and increase glucose uptake by muscle and adipose tissue. m, increase; k, decrease.

ethanolic extract of saffron stigma to mildly diabetic and severely diabetic rats resulted in 33% and 27% increase in the serum insulin level, respectively (Mohajeri et al., 2008). In a study by Kianbakht and Hajiaghaee (2011), the administration of methanolic extract of saffron, crocin, and safranal to diabetic rats could double the level of serum insulin. In this study, the effect of saffron and its active compounds on insulin level was the same as that of glibenclamide. Histological examination of the pancreas of saffron-treated diabetic rats revealed the restoration of normal pancreatic architecture and the improvement of the islet structure (Ali et al., 2016; Elgazar et al., 2013). Consistent with this finding, safranal was reported to reduce the oxidative stress and inflammation in the pancreas tissue of T2D rats (Hazman and Ovalı, 2015). It has been shown that safranal and alcoholic extracts of saffron stimulated glucose uptake in myocytes and adipocytes by increasing the expression and translocation of glucose transporter 4 (Dehghan et al., 2016; Kang et al., 2012; Maeda et al., 2014). Activation of mitogen-activated protein kinases and AMP-activated protein kinase pathways play a major role in the effects of saffron on insulin sensitivity and glucose uptake in myocytes (Dehghan et al., 2016; Kang et al., 2012). Also, safranal can inhibit protein tyrosine phosphatase 1B, which suppress insulin signaling through dephosphorylation of the insulin receptor, and therefore prompts a ligand-independent stimulation of insulin signaling (Maeda et al., 2014). In addition, crocetin, a deglycosylated crocin derivative, was found to decrease TNF-α and increase adiponectin expression in adipose tissue. These favorable impacts of crocetin have been suggested to be involved in the improvement of insulin sensitivity observed in crocetin-treated rats (Xi et al., 2007a,b). Studies have shown that crocin can inhibit the activity of α-amylase and α-glucosidases (Xiao-Ping et al., 2010). Inhibition of these carbohydrate-hydrolyzing enzymes delays the liberation of glucose from nutritional complex carbohydrates and avoids the sharp rise in the level of postprandial serum glucose.

31.3.3 Effects of saffron on diabetic complications 31.3.3.1 Diabetic neuropathy Neuropathy is developed in more than half of all diabetic patients and involves the dysfunction of the autonomic system (e.g., orthostatic hypotension); a defect in sensory perception (e.g., paresthesias, hyperalgesia, and allodynia); and the dysfunction of motor neurons (Brownlee, 2005). Results of studies on diabetic animals confirm the benefits of saffron and its active compounds on diabetic neuropathy (Fig. 31.2). Crocin and safranal can ameliorate neuropathic pain (such as cold allodynia and hyperalgesia) in the streptozotocin (STZ) model of T1D (Farshid and Tamaddonfard, 2015; Hosseinzadeh et al., 2009). It has been reported that the protective effect of crocin and safranal against the sciatic nerve injury is as effective as insulin (Farshid and Tamaddonfard, 2015). Saffron compounds also protects the hippocampus, cerebrum, and cerebellum by inhibiting against histopathological changes (Altinoz et al., 2014a; Samarghandian et al.,

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FIGURE 31.2 Protective effects of saffron and its compounds on organs highly vulnerable to diabetic injury. m, increase; k, decrease.

2014b; Tamaddonfard et al., 2013). Oxidative stress and neuroinflammation are among the main causes of the development and progression of diabetic neuropathy (Sandireddy et al., 2014). It is well known that saffron compounds are able to decrease oxidative stress in different parts of the nervous system such as brain cortex and sciatic nerve (Farshid and Tamaddonfard, 2015; Mousavi et al., 2010; Samarghandian et al., 2014b; Tamaddonfard et al., 2013). The hippocampus is an important area of the brain due to its involvement in learning and memory processes (Eichenbaum, 2004). Individuals with diabetes have a reduced hippocampus volume as a result of apoptosis-induced neuronal loss (Gold et al., 2007; Li et al., 2002). Chronic treatment with crocin at doses of 15
60 mg kg21 was shown to inhibit hippocampal neuronal loss and to improve learning and memory performance in diabetic animals (Ahmadi et al., 2017; Tamaddonfard et al., 2013).

31.3.3.2 Diabetic nephropathy Nephropathy is one of the main microvascular complications of diabetes. Experimental and clinical studies have demonstrated that saffron compounds improve the function of kidney in both normal and diabetic subjects (Ahmadi et al., 2017; Derakhshanfar et al., 2015; Elgazar et al., 2013; Hazman and Bozkurt, 2015; Karafakıo˘glu et al., 2017; Milajerdi

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et al., 2017; Rajaei et al., 2013; Shirali et al., 2013). Ethanolic extract of saffron has been shown to the serum concentrations of urea and creatinine in alloxan-induced diabetic rats (Elgazar et al., 2013). A similar effect was also produced by administration of safranal (Hazman and Bozkurt, 2015). Another compound of saffron, crocin, was shown to reduce microalbuminuria in STZ-induced diabetic rats (Shirali et al., 2013). Safranal and crocin inhibit renal tissue damage through their antiinflammatory and antioxidant properties (Hazman and Bozkurt, 2015; Rajaei et al., 2013). In a double-blind clinical trial on type-2 diabetic patients, administration of a hydroalcoholic extract of saffron stigma (15 mg, twice a day, for 8 weeks) decreased the level of urea nitrogen in the blood (Milajerdi et al., 2017).

31.3.3.3 Cardiovascular complications Diabetes increases the risk of developing cardiovascular disease (Fowler, 2008). Saffron is suggested as one of the best supplements for cardiac health due to the antiinflammatory, antioxidant, and hypolipidemic effects of its compounds (Kamalipour and Akhondzadeh, 2011). Treatment of diabetic rats with crocin decreases lipid peroxidation and increases the activity of antioxidant enzymes in the heart (Altinoz et al., 2015; Farshid et al., 2016; Ghorbanzadeh et al., 2016). Crocin also improves the effects of STZ on ECG (by decreasing heart rate and increasing QT interval and T wave amplitude) and reduces the serum levels of biochemical markers of myocardial damage such as creatine kinase myocardial band (CK-MB) and lactate dehydrogenase (LDH). The combined administration of crocin and insulin produces more effects on serum activities of LDH and CK-MB and induces more antioxidant activity on heart tissue when compared to an administration of each chemical by itself (Farshid et al., 2016). Histopathological examinations revealed that crocin can improve tissue damage in the heart: it decreases interstitial edema, fibroblastic proliferation, hemorrhagic area, inflammation, and myocardial vacuolization and degeneration (Altinoz et al., 2015; Farshid et al., 2016). Additionally, crocin could decrease the levels of inflammatory cytokines TNF-α and IL-6 in the aorta of diabetic rats (Samarghandian et al., 2016). Increased production of AGEs was shown to play an important role in the development of vascular complications (Yamagishi et al., 2015). Crocetin is able to inhibit AGE-induced endothelial cell apoptosis and prevent adhesion of leukocytes to vascular cells (Xiang et al., 2006a,b). Many patients with T1D and T2D show increased levels of TG and LDL as well as a reduced level of HDL. It is well documented that dyslipidemia is one of the main risk factors for developing atherosclerosis in diabetic patients. Although patients with diabetic dyslipidemia receive different hypolipidemic drugs, such as statins, fibrates, etc., a large number of patients do not reach the LDL baseline of ,70 mg dL21 (Dake and Sora, 2016). Therefore, the use of natural hypolipidemic agents may prove beneficial in the prevention and or treatment of cardiovascular diseases. The beneficial effects of saffron and its active derivatives on the lipid profile of diabetic animals have been shown in several experimental studies (Table 31.1). A decrease in serum TG, LDL, and very low-density lipoprotein was observed following treatment with saffron extract, safranal, and crocin (Ali et al., 2016; Altinoz et al., 2014a, 2015; Bajerska et al., 2013; Dehghan et al., 2016; Elgazar et al., 2013; Hemmati et al., 2015a,b; Jin et al., 2009; Konstantopoulos et al., 2017; Lari et al., 2014; Samarghandian et al., 2013, 2014b, 2016; Shirali et al., 2012, 2013). Current data regarding the effect of saffron stigma on TC and HDL are conflicting. Some studies have reported an increase in the HDL level and a decrease in the TC level following administration of saffron (Ali et al., 2016; Altinoz et al., 2014a, 2015; Elgazar et al., 2013; Jin et al., 2009; Samarghandian et al., 2013, 2014b, 2016; Shirali et al., 2012, 2013). However, other studies indicated that saffron stigma had no significant effects on HDL and/or TC in diabetic animals (Bajerska et al., 2013; Dehghan et al., 2016; Hemmati et al., 2015a,b; Konstantopoulos et al., 2017).

31.3.3.4 Diabetic retinopathy Diabetic retinopathy is a common microvascular complication of diabetes that can ultimately cause loss of vision. The pathogenesis of this disease is very complex, involving different factors such as hyperglycemia (an increase in the formation of AGEs, activation of protein kinase C, etc.); hyperlipidemia (an excess of ketone bodies, oxidized fatty acids, etc.); growth factors (including insulin-like growth factor, endothelins, etc.); neurodegeneration (such as glutamate neurotoxicity); and inflammation (involving interleukins, TNF-α, etc.) (Ahsan, 2015). It has been suggested that saffron derivatives may facilitate retinal function recovery after different pathologic conditions including ischemic injury, agerelated macular degeneration, and diabetic retinopathy (Bhandari, 2015; Ferna´ndez-Sa´nchez et al., 2012; Guo et al., 2012; Lv et al., 2016; Qi et al., 2013; Xuan et al., 1999; Yang et al., 2017). Ferna´ndez-Sa´nchez et al. (2012) reported that saffron derivatives attenuates retinal degeneration in a rat model of autosomal dominant retinitis pigmentosa. The study showed that safranal conserves a capillary network of the retina and preserves both the number and morphology of photoreceptors. Qi et al. (2013) showed that crocin protects retinal ganglion cells against ischemia/reperfusioninduced apoptosis. Crocin also protects the ganglion cells against H2O2-induced apoptosis through the decrease of

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reactive oxygen species generation and downregulation of proapoptotic protein expression (Lv et al., 2016). It has been suggested that high levels of glucose and free fatty acids may cause the overactivation of microglial cells, which subsequently cause neurotoxicity and apoptosis of retinal ganglion cells. Crocin was shown to prevent the oxidative stress and proinflammatory response induced by high glucose and free fatty acids (Yang et al., 2017). In another study, crocin could prevent the AGE-induced apoptosis of retinal microvascular endothelial cells through inhibiting ROS production and decreasing TNF-α level (Guo et al., 2012).

31.3.3.5 Liver damage Diabetes is a risk factor for developing chronic liver damage and the progression of nonalcoholic steatohepatitis to cirrhosis (Amarapurkar and Das, 2002). It has been suggested that some complications of diabetes (e.g., neuropathy and retinopathy) are associated with the intensity of activities of liver enzymes, independent of BMI, alcohol intake, serum lipids, and the level of HbA1c (Arkkila et al., 2001). Oxidative stress is one of the main biochemical triggers for diabetic liver damage (Kakkar et al., 1998; Lucchesi et al., 2013). An increasing line of evidence confirms that saffron compounds may be effective in preventing diabetic liver injury (Ali et al., 2016; Altinoz et al., 2014b; Elgazar et al., 2013; Konstantopoulos et al., 2017; Kianbakht and Hajiaghaee, 2011; Milajerdi et al., 2017; Peeri et al., 2012; Rahbani et al., 2011, 2012). Several studies demonstrated that saffron and different extracts prepared from its stigma reduces serum levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and bilirubin in diabetic rats (Ali et al., 2016; Elgazar et al., 2013; Konstantopoulos et al., 2017; Peeri et al., 2012; Rahbani et al., 2011, 2012). These effects were accompanied by an increase in hepatic antioxidant activities of glutathione peroxidase, superoxide dismutase, and catalase (Peeri et al., 2012; Rahbani et al., 2011). Saffron is also able to improve histological changes of the liver by decreasing fat accumulation, sinusoid dilations, and necrosis (Konstantopoulos et al., 2017; Peeri et al., 2012; Rahbani et al., 2011). The beneficial effects of saffron extracts on hepatic enzymes and oxidative stress may be due to the presence of crocin (Altinoz et al., 2014b; Rajaei et al., 2013). In spite of the aforementioned positive results regarding hepatic enzymes, some studies reported that saffron extract, crocin, and safranal did not have significant effects on the blood levels of AST and ALT (Kianbakht and Hajiaghaee, 2011; Milajerdi et al., 2017). It seems that such positive effects are only observed when the level of hepatic enzymes is significantly high in diabetic subjects.

31.3.3.6 Other complications of diabetes A number of studies suggest the benefits of saffron compounds in the management of other complications associated with diabetes, such as erectile dysfunction, gastropathy, and respiratory distress (Kianbakht and Mozaffari, 2009; Mohammadzadeh-Moghadam et al., 2015; Shamsa et al., 2009). In a randomized double-blind, placebo-controlled trial, a topical formulation of saffron could improve erectile dysfunction in diabetic patients (Mohammadzadeh-Moghadam et al., 2015). In another study, saffron showed similar positive action (increased the number and duration of erectile events) in nondiabetic patients (Shamsa et al., 2009). In spite of these positive reports, in one open-label randomized crossover study comparing the efficacy of sildenafil and saffron for treating erectile dysfunction, no significant improvements were observed with regard to erectile dysfunction in nondiabetic nor controlled diabetic (HbA1c # 7) patients (Safarinejad et al., 2010). Therefore, further well-designed clinical trials are necessary to confirm the advantages of saffron for erectile dysfunction in diabetic patients. It has been reported that saffron extract, safranal, and crocin could prevent the gastric lesions induced by indomethacin in diabetic rats (Kianbakht and Mozaffari, 2009). The significance of this finding is that the gastric mucosa of diabetic subjects is more vulnerable to peptic ulcer bleeding (Peng et al., 2013). Administration of safranal for 4 weeks has been shown to decrease malondialdehyde and nitric oxide. The same 4-week administration was also found to increase reduced glutathione, superoxide dismutase, and catalase in the bronchoalveolar lavage fluid and lung tissue of STZ-induced diabetic rats. This suggests that safranal may be effective at preventing lung distress in diabetes by ameliorating oxidative damage (Farahmand and Samarghandian, 2012; Samarghandian et al., 2014a).

31.4

Conclusion

Pharmacological studies have confirmed the effectiveness of saffron in managing some complications of obesity and diabetes. In obesity, it reduces food intake, weight gain, inflammatory possesses, and oxidative stress, and may reverse unfavorable changes in the levels of adipocytokines. In diabetes, saffron derivatives have antihyperglycemic properties

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and are able to prevent or treat associated complications such as nephropathy, neuropathy, retinopathy, liver damage, and cardiovascular disorders. Although this plant and its active derivatives have shown multiple useful effects in the treatment of metabolic disorders, clinical evidence is still limited in this regard, and further well-designed clinical trials are required to evaluate the advantages and limitations of saffron for managing obesity and diabetes.

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518. Lari, P., Rashedinia, M., Abnous, K., Hosseinzadeh, H., 2014. Crocin improves lipid dysregulation in subacute diazinon exposure through ERK1/2 pathway in rat liver. Drug Res. 64, 301
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231. Lorenzati, B., Zucco, C., Miglietta, S., Lamberti, F., Bruno, G., 2010. Oral hypoglycemic drugs: pathophysiological basis of their mechanism of action oral hypoglycemic drugs: pathophysiological basis of their mechanism of action. Pharmaceuticals 3, 3005
3020.

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Lucchesi, A.N., Freitas, N.T.D., Cassettari, L.L., Marques, S.F.G., Spadella, C.T., 2013. Diabetes mellitus triggers oxidative stress in the liver of alloxan-treated rats: a mechanism for diabetic chronic liver disease. Acta Cir. Bras. 28, 502
508. Lv, B., Chen, T., Xu, Z., Huo, F., Wei, Y., Yang, X., 2016. Crocin protects retinal ganglion cells against H2O2-induced damage through the mitochondrial pathway and activation of NF-κB. Int. J. Mol. Med. 37, 225
232. Maeda, A., Kai, K., Ishii, M., Ishii, T., Akagawa, M., 2014. Safranal, a novel protein tyrosine phosphatase 1B inhibitor, activates insulin signaling in C2C12 myotubes and improves glucose tolerance in diabetic KK-Ay mice. Mol. Nutr. Food Res. 58, 1177
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308. Mashmoul, M., Azlan, A., Yusof, B.N.M., Khaza’ai, H., Mohtarrudin, N., Boroushaki, M.T., 2014. Effects of saffron extract and crocin on anthropometrical, nutritional and lipid profile parameters of rats fed a high fat diet. J. Funct. Foods 8, 180
187. Mashmoul, M., Azlan, A., Mohtarrudin, N., Nisak, B., Yusof, M., Khaza’ai, H., 2017. Saffron extract and crocin reduced biomarkers associated with obesity in rats fed a high-fat diet. Malays. J. Nutr. 23, 117
127. Milajerdi, A., Jazayeri, S., Bitarafan, V., Hashemzadeh, N., Shirzadi, E., Derakhshan, Z., et al., 2017. The effect of saffron (Crocus sativus L.) hydroalcoholic extract on liver and renal functions in type 2 diabetic patients: a double-blinded randomized and placebo control trial. J. Nutr. Intermed. Metab. 9, 6
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Chapter 32

Anticancer properties of saffron Jalil Tavakol-Afshari1, Mohammad-Hossein Boskabady2,3 and Roshanak Salari4 1

Immunology Research group, Buali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran, 2Neurogenic Inflammation Research

Center, Mashhad University of Medical Sciences, Mashhad, Iran, 3Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 4Department of Pharmaceutical Sciences in Persian Medicine, School of Persian and Complementary Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 32.1 32.2 32.3 32.4 32.5 32.6 32.7

Introduction Lung cancer Pancreatic cancer Colorectal cancer Breast cancer Leukemia Hepatic cancer

32.1

485 486 487 487 487 488 489

32.8 Cervical cancer 32.9 Skin cancer 32.10 Prostate cancer 32.11 Gastric cancer 32.12 Conclusion References

489 490 490 490 491 491

Introduction

Cancer is one of the major noncommunicable health threats to humans, claiming millions of lives each year. It refers to abnormal growth of cells that spread and metastasize to more distant parts from the origin through uncontrolled cell division. Chemoprevention is the most widely used therapeutic option to block cancer development apart from other emerging procedures like radiotherapy, photodynamic therapy, catalytic therapy, and surgical intervention in some cases. To date, no ideal approach has been found to obtain satisfactory effects against cancer. An appropriate strategy for cancer prevention or treatment could be a combined approach, including the application of synthetic or natural agents to inhibit cancer development. Evidence shows that plants, such as vegetables, spices, and herbs, have anticancer chemoprevention and could be used in new drug development. Natural remedies have more advantages, including little or no toxicity and low cost (Mallath et al., 2014; Vineis and Wild, 2014). Saffron is the dried stigmas of Crocus sativus L. and belongs to the family of Iridaceae, the line of Liliaceae, and is mainly grown in the Mediterranean Sea through Iran to India, Tibet, and other regions in China (Champalal et al., 2011). The main reason saffron is expensive is because it is still cultivated and harvested, as it has been for millennia, by hand. In traditional medicine, saffron has been used to treat many diseases. It is also valued as a food additive for tasting, flavoring, and coloring, along with its therapeutic properties. Based on chemical analyses of the dry stigma of saffron extracts, carotenoids, namely, crocin and crocetin and the monoterpene aldehydes picrocrocin and safranal, are the most important active carotenoid secondary metabolites of saffron. Crocin with chemical formula of C44H64O24 and molecular weight 976.96, is a hydrophilic carotenoid (8´-diapocarotene-8, 8´-dioic acid), and constitutes approximately 6%–16% of saffron’s total dry matter depending on the variety, growing conditions, and processing methods. This is the diester formed from the disaccharide gentiobiose and the dicarboxylic acid crocetin. The deep red color of crocin produces the color of saffron. Crocin 1 (or -crocin), a digentiobioside, is the most abundant crocin with high solubility attributed to these sugar moieties. Crocin is widely used as a natural food colorant. In addition to crocin, saffron contains crocetin as a free agent and small amounts of the pigment anthocianin, -carotene, -carotene, and zegxantin. Crocetin, with elementary composition (C20H24O4), melting point 285˚C, and molecular weight 328.4, is an amphiphilic low-molecular-weight natural carotenoid (8, 8´-diapo-8, 8´-carotenoic acid) and consists of a C-20 carbon chain with Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00033-2 © 2020 Elsevier Inc. All rights reserved.

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seven double bonds and a carboxylic acid group at each end of the molecule. This compound is present in the central core of crocin and is responsible for the color of saffron, constituting approximately 14% of saffron’s total dry matter depending on the variety, growing conditions, and processing methods. It is soluble in organic bases and slightly soluble in aqueous solution (20 M at pH 8.0). Picocrocin with elementary composition (C16H26O7) and molecular weight 330.37 g mol1 is the main bitter crystalline terpene-glucoside of saffron (Gutheil et al., 2012). The actual taste of saffron is derived primarily from picrocrocin, which is the second most abundant component (by weight), accounting for approximately 1%–13% of saffron’s dry matter. The action of -glucosidase on picrocrocin liberates the aglycone4hydroxy-2, 6, 6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC, C10H16O2), which is transformed to safranal by dehydration during the drying process of the plant material. Natural deglycosylation of picrocrocin will yield another important aroma factor, safranal, (C10H14O), which is comprised of about 60% of the volatile components of saffron. Dehydration is not only important to the preservation of saffron but is actually critical in the release of safranal from picrocrocin via enzymatic activity, the reaction yielding D-glucose and safranal, the latter being the volatile oil in saffron. Safranal, with elementary composition (C10H14O) and molecular weight 150.21 g mol1, is the major volatile oil responsible for the aroma (Rezaee and Hosseinzadeh, 2013). Toxicological studies have shown that the toxicity of saffron is quite low and oral LD50 of saffron in animal was 20.7 g kg1 administrated as a decoction. It has been demonstrated that oral administration of saffron extract at doses from 0.1 to 5 g kg1 was nontoxic in mice. Ames/Salmonella test system was revealed that crocin and dimethyl-crocetin isolated from saffron were nonmutagenic and nontoxic. Saffron and its constituents beneficially affect cancer, heart disease, metabolic disorders, and cognitive disorders by different mechanisms: 1. Prevents carcinogenesis: Saffron and its constituents start working long before a cell undergoes transformation into a malignant cancer cell. The first step in cancer development, or carcinogenesis, is some kind of trigger that initiates malignant transformation. This may be an environmental toxin, a stray oxygen radical, or invasion with certain viruses. Saffron components have been shown to help prevent the carcinogenesis caused by each of these triggers. Saffron extracts and specific components have also been shown to potently prevent DNA damage caused by free radicals, radiation, and inflammation, thereby reducing the risk of new cancer formation. 2. Inhibits the rapid spread of cancer cells: Once a cell has been triggered to become malignant, it then proliferates, or reproduces rapidly and without normal controls, to produce a developing tumor. Studies show that saffron is able to suppress and in some cases reverse the proliferation of certain human cancer cells in culture. 3. Triggers programmed cell death: Another important way in which developing cancer cells can be stopped in their tracks is through the mechanism known as apoptosis, or programmed cell death. All normal body cells contain a genetic program that induces the cell to die under specific conditions. This is a vital means of removing damaged cells and preventing overgrowth of normal tissues. Cancer cells, however, typically lose their responsiveness to the apoptosis signal, effectively becoming “immortal,” and hence, deadly. Saffron has been shown to trigger apoptosis in a variety of cancer cell lines, which is seen as an essential component of any cancer-control or cancer-prevention method. In fact, all three major components of saffron, crocin, crocetin, and safranal, have shown powerful apoptosis-inducing properties. 4. Prevents metastasis: If a cancer cell survives attempts to squash it by blocking proliferation or apoptosis, it may go on to produce specialized molecules that help it degrade the protein matrix between healthy cells, allowing it to invade otherwise-healthy tissue. This is how cancers spread locally, and it is also a major mechanism in metastasis, the spread of malignant cells throughout the body. The saffron constituent crocetin has been shown to downregulate production of one such protein-degradation molecular type, known as matrix metalloproteinase. This action has been shown to prevent breast cancer cells from penetrating and invading both local tissues and those in other parts of the body by metastasis. 5. Blocks angiogenesis: Still another means by which growing tumors are able to thrive is through the induction of new blood vessel growth, a process known as angiogenesis. Considerable scientific effort has been devoted to developing drugs that can block angiogenesis, thereby starving a developing tumor of the nutrients and oxygen it needs to sustain growth. The studies by Kumar et al. (2011) and Samarghandian and Borji (2014) support the use of saffron extracts in reducing levels of a vital signaling molecule called vascular endothelial growth factor, which markedly reduces new blood vessel formation in the tumor mass.

32.2

Lung cancer

Lung cancer is the number one cause of cancer deaths in both men and women worldwide. Lung cancer, also known as lung carcinoma, is a malignant lung tumor characterized by uncontrolled cell growth in tissues of the lung. This growth

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can spread beyond the lung by the process of metastasis into nearby tissue or other parts of the body. In two studies, the induction of cytotoxicity and apoptosis of the ethanolic extract of saffron in human cancer alveolar basal epithelial cells (A549) was evaluated in vitro. Results from both studies showed that saffron, in a concentration and timedependent manner, could reduce the cell viability and proliferation. The apoptotic effects of the saffron extract in a human lung cancer-derived cell line could be considered as a potential chemotherapeutic agent in lung cancer. It has been shown that crocetin (at doses of 100–150 g mL1) exhibits inhibitory effects on nucleic acid synthesis and colony formation of A549 (lung carcinoma) and VA13 (transformed SV-40 of fetal lung fibroblasts) cells. The effects of crocetin were evaluated against lung cancer-bearing mice in both the pre- and postinitiation periods. Crocin treatment normalized the raised levels of lipid peroxidation and marker enzymes markedly in carcinogen administered animals. It also raised the activities of the antioxidants and glutathione-metabolizing enzymes. The pathological changes seen in cancerous animals were notably normalized by crocetin administration. The anticancer activity of the aqueous extract of saffron could be attributed partly to its inhibition of the cell proliferation and induction of apoptosis in cancer cells through caspase-dependent pathways activation (Samarghandian et al., 2010, 2011, 2013).

32.3

Pancreatic cancer

Pancreatic cancer is an aggressive type of cancer with few symptoms until the cancer is advanced. A median survival of 6 months and a dismal 5-year survival rate of 3%–5% is seen with this cancer (Iovanna et al., 2012; Ling et al., 2014). A series of experiments were conducted to systematically establish whether crocetin significantly affects pancreatic cancer growth both in vitro and/or in vivo (Boreddy and Srivastava, 2013). A study in 2009 investigated anticancer potential of crocetin on pancreatic cancer using different pancreatic cancer cells as well as a xenograft athymic mice model. Crocetin at doses of 50–200 mol L1 for 72 hours inhibited proliferation of MIA-PaCa-2, BxPC3, Capan1, and Ascpc1 cells. The study also indicated that DNA synthesis was inhibited in pancreatic cancer cells in the presence of crocetin (Dhar et al., 2009). Subsequently, cell cycle proteins were evaluated. Cdc-2, Cdc-25C, Cyclin-B1, and epidermal growth factor receptor were modified significantly by crocetin. In an in vivo study, MIA-PaCa-2 as highly aggressive cells than other pancreatic cancer cells were injected into the right hind leg of the athymic nude mice and crocetin was given orally after the development of a palpable tumor. The in vivo results demonstrated a significant decrease in tumor growth along with reduction of proliferation as estimated by proliferating cell nuclear antigen and epidermal growth factor receptor expression in the crocetin-treated animals compared with the controls. The experiment by Bakshi et al. (2010) demonstrated that crocetin along with low doses of paclitaxel or cisplatin can inhibit proliferation and stimulate apoptosis of pancreatic cancer cells. Therefore, these studies provide evidence that crocetin can act safely along with the common chemotherapy drugs as an antitumorigenic on pancreatic cancer.

32.4

Colorectal cancer

Colorectal cancer is one of the most common cancers in the world. It includes 9% of all cancers. This cancer is the second most common cancer. The incidence of colorectal cancer has been a great increase in the world. Colorectal cancer occurs when some of the cells that line the colon or the rectum become abnormal and grow out of control (Haraldsdottir et al., 2014). Since colon cancer takes 15–20 years to transition from premalignant stages to cancer, natural dietary treatments are a simple protective measure against this disease. Aung et al. (2007) reported that aqueous extract of saffron and its major constituent, crocin, significantly inhibited the growth of colorectal cancer cells while not affecting normal cells. They demonstrated major concentration-related inhibition effects of the extract on three colorectal cancer cell lines (HCT-116, SW-480, and HT-29) compared to that of nonsmall cell lung cancer cells by MTS assay. Results from this study showed that saffron extract and its major constituent, crocin, significantly inhibited the growth of colorectal cancer cells while not affecting normal cells (Aung et al, 2007). Another study investigated the p53-dependency of saffron’s mechanism of action in two p53 isogenic HCT116 cell lines (HCT wild type and HCT p53/) and showed induction of DNA damage and apoptosis in both cell lines. However, autophagy delayed the induction of apoptosis in HCT116 p53/ cells (Bajbouj et al., 2012).

32.5

Breast cancer

Breast cancer is a cancer that forms in the cells of the breasts and is the most common cancer among women. Breast cancer can occur in both men and women, but it’s far more common in women. Chemotherapy and surgery are common

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treatments against breast cancer. However, some breast cancers are not killed by chemotherapy (Advani and MorenoAspitia, 2014). It has been reported that crocetin and its analogs inhibited breast cancer cell proliferation and cell viability. In this study, a dose-dependent inhibition of MDA-MB-231 and MCF breast cancer cells proliferation was observed in the presence of crocetin. This effect was independent of the estrogen receptors function. Hence, the antitumor mechanism of crocetin is independent of hormone regulation. Another study also investigated the proapoptotic effects of crocetin on MCF7 cells, indicating that crocetin leads to induction of a caspase-dependent pathway through elevation of the BAX protein expression. MCF7/VCR cell line is resistant to a chemo drug called vincristine. However, treating these cells with saffron caused them to die by activating two different mechanisms; when both saffron and vincristine were given at the same time, the saffron made the MCF7/VCR cells vulnerable to the vincristine. Other studies reported that saffron and crocetin induced apoptosis on human breast cancer cell (MCF-7) via p53-mediated stimulation of apoptosis. The results indicated that caspase-dependent pathway was induced by saffron in MCF-7 cells and Bax protein expression was also increased in saffron-treated cells. In another study, the potential of the ethanolic extract of saffron to induce antiproliferative and cytotoxic effects in cultured carcinomic human alveolar basal epithelial cells (MCF-7) in comparison with nonmalignant (L929) cells was studied. The results showed that even higher concentrations of saffron is safe for L929, but its proapoptotic effects in a lung cancer-derived cell line could be considered as a potential chemotherapeutic agent in lung cancer. Parallel to this study, the effect of crocin and its nanoliposomal was examined on MCF-7 cells by measuring apoptotic cell via flow cytometry method. Results indicated that crocin and its liposomal form induced a sub-G1 peak in flow cytometry histogram of treated cells, indicating apoptosis is involved in this toxicity. Liposomal encapsulation enhances apoptogenic effects of crocin on cancerous cells. It might be concluded that crocin could cause cell death in MCF-7 cells, in which liposomal encapsulation improved its cytotoxic effects. MCF-7 and MDA-MB-231 breast cancer cells showed concentration-dependent inhibition of proliferation by crocetin and this effect was independent of estrogen receptor. A study on the effect of crocetin on breast cancer cells MCF-7 and invasive MDA-MB-231 indicted that crocetin, the main metabolite of crocins, inhibits MDA-MB-231 cell invasiveness via downregulation of MMP expression. Thus, these studies suggested that crocetin can be used as a chemopreventive agent in breast cancer (Chryssanthi et al., 2011; Singh, 2009).

32.6

Leukemia

Leukemia is a cancer of the blood or bone marrow. Bone marrow produces blood cells. Leukemia can happen when there is a problem with the production of blood cells. It usually affects the leukocytes, or white blood cells. It is most likely to affect people over the age of 55 years, but it is also the most common cancer in those aged under 15 years. Most studies on the anticarcinogenic effect of saffron have been focused on crocin as an important anticancer compound. In several studies, inhibition of growth of human chronic myelogenous leukaemia K562 and promyelocytic leukaemia HL-60 cells by dimethylcrocetin, crocetin, and crocin, with 50% inhibition (ID50) reached at concentrations of 0.8, 2, and 2 mM, respectively. Cytotoxicity of dimethylcrocetin and crocin to various tumor cell lines (L1210 leukemia and P388 leukemia) has been reported, with concentrations producing 50% cytotoxicity ranging from 7 to 30 mg mL1 for dimethyl-crocetin and from 11 to 39 mg mL1 for crocin. These authors detected significant inhibition in the synthesis of nucleic acids, and suggested that dimethyl-crocetin could disrupt DNA-protein interactions (e.g., toposiomerases II) important for cellular DNA synthesis. Oral administration of saffron extract significantly inhibited genotoxicity induced by cisplatin, mitomycin-C, and urethane in the mouse bone marrow micronucleus test. Reseachers assessed the effects of aqueous extracts of saffron (composed mainly of carotenoids) in Swiss albino mice, and suggested that pretreatment with saffron can significantly inhibit the genotoxicity of cisplatin, cyclophosphamide, mitomycin, and urethane. In an experiment to evaluate its protective effect on cisplatininduced toxicity in rats (3 mg kg1 body wt.), results showed that treatment of animals with cysteine (20 mg kg1 body wt.) together with saffron extract (50 mg kg1 body wt.) significantly reduced the toxic effects caused by cisplatin, such as nephrotoxicity and changes in enzyme activity. The results of the study conducted by Sun et al. demonstrated that crocin suppressed HL-60 cell proliferation and stimulated apoptosis and cell cycle arrest at G0/G1 phase, in a concentration- and timedependent manner. Furthermore, crocin reduced the tumor weight and size of HL-60 xenografts in nude mice, suppressed Bcl-2 expression, and enhanced Bax expression in xenografts. Cytotoxicity experiments conducted by Geromichalos et al. (2014) demonstrated that safranal (SFR) and crocin mediate cytotoxic response to K562 cells (human chronic myelogenous leukemia cells). SFR and to a lesser extent imatinib mesylate (used in the treatment of human chronic myelogenous leukemia) suppressed the expression of Bcr-Abl gene expressing Bcr-Abl protein tyrosine kinase activity in in vitro studies. Additionally, in silico molecular docking experiments showed that SFR can be attached to Bcr-Abl protein, positioned within the protein’s binding cavity at the same place with imatinib mesylate. Evaluation of the effects of crocin on human T-cell leukemia cell line, MOLT-4, demonstrated that crocin exhibited mild cytotoxic effects on a leukemia cell line, which might be mediated through the increase of DNA fragmentation (Geromichalos et al., 2014; Rezaee et al., 2013; Sun et al., 2013).

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Hepatic cancer

Liver cancer, also known as hepatic cancer and primary hepatic cancer, is cancer that starts in the liver. In this condition, normal cells in the liver become abnormal in appearance and behavior. The cancer cells can then become destructive to adjacent normal tissues, and can spread both to other areas of the liver and to organs outside the liver (Lim and Torresi, 2014). Several investigations show that saffron exerts an important chemopreventive effect against liver cancer due to the inhibition of cell proliferation and induction of apoptosis. In vitro experiments on HepG2 cells showed inhibition of nuclear factor-kappa B activation, increased cleavage of caspase-3, as well as DNA damage and cell cycle arrest upon saffron treatment (Amin et al., 2011). In addition, the telomerase activity of HepG2 cells reduces after treatment with crocin, which is probably caused by downregulation of the expression of the catalytic subunit of the enzyme (Noureini and Wink, 2012). The results of another study showed that saffron exerts a significant chemopreventive effect against diethylnitrosamine (DEN)-induced liver cancer (HepG2) through inhibition of cell proliferation via induction apoptosis, modulating oxidative damage and suppressing inflammatory response. One study focused on the antiproliferative effects of crocin on HepG2 cells. In this study melting temperature of a synthetic telomeric oligonucleotide was measured in the presence of crocin by using FRET analysis to examine two probable mechanisms of crocin inhibition: interaction of crocin with telomeric quadruplex sequences and downregulation of hTERT expression. Data indicated that telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by downregulation of the expression of the catalytic subunit of the enzyme. The cytotoxicity and DNA-adduct formation of rat liver microsomes activated by aflatoxin B1 (AFB1) in the C3H10T1/2 fibroblast cells were significantly inhibited by pretreatment of crocetin. Crocetin treatment resulted in a decrease in AFB1-DNA adduct formation in vitro that suggested the protective effect of crocetin on the AFB1 cytotoxity due to the elevation of cytosolic glutathione (GSH) following the activities of GSH-S-transferase (GST) formation as cellular defense mechanisms. Crocetin pretreatment in rats protected hepatic AFB1-induced hepatic damage and AFB1-DNA adduct formation due to the elevation hepatic GSH, activities of GST, and glutathione peroxidase (GSH-Px). According to one study, significant suppression of AFB1induced hepatotoxic lesions was observed as indicated by reduction of activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transpeptidase by crocetin in rats. Inhibitory effect of crocetin on benzo[a] pyrene-induced genotoxicity and neoplastic transformation in C3H1OT1/2 cells was due to a mechanism that increased the activity of GSH and reduced the formation of benzo[a] pyrene-DNA adducts. Crocetin also inhibited the formation of malondialdehyde, a marker for lipid peroxidation, induced by reactive oxygen species (ROS) generated by the activity of xanthine oxidase in primary hepatocytes and protected against oxidative damage. Therefore, these studies indicated that crocetin displayed protective action against the ROS due to direct scavenging that inhibited free radical production following neoplastic transformation. Saffron significantly reduced the DENinduced increase in the number and the incidence of hepatic dyschromatic nodules in rats. Saffron also diminished the number and the area of placental glutathione S-transferase-positive foci in livers of DEN-treated rats. Moreover, saffron counteracted DEN-induced oxidative stress in rats as evaluated by restitution of superoxide dismutase, catalase, and glutathione-S-transferase levels, and decreased myeloperoxidase activity, malondialdehyde, and protein carbonyl formation in liver. The results of immunohistochemical staining of rat liver demonstrated that saffron inhibited the DEN-mediated elevations in the numbers of cells positive for cyclooxygenase 2, inducible nitric oxide synthase, nuclear factor-kappa B p-65, and phosphorylated tumor necrosis factor receptor. This study also confirms some evidence that saffron protects rat liver from cancer via modulating oxidative damage and suppressing inflammatory response (Amin et al., 2011; Costantini et al., 2014; Noureini and Wink, 2012).

32.8

Cervical cancer

Cervical cancer is a cancer arising from the cervix. It is due to the abnormal growth of cells that have the ability to invade or spread to other parts of the body. Cancer of the cervix often has no symptoms in its early stages (Sasieni, 2006). Several data reported that saffron may cause significant reduction in the colony formation and DNA and RNA synthesis at doses of 1200 g mL1 in HeLa cells (derived from a cervical epitheloid carcinoma). Crocetin inhibits DNAdependent RNA polymerase II and RNA synthesis. UV spectroscopy showed an interaction between crocetin and tRNA. In another study, ROS did not play an effective role in the cytotoxic effect of saffron extract on HeLa cell lines, in which apoptosis or programmed cell death plays an important role. The effect of crocin and its nanoliposomal was examined on HeLa cells by measuring apoptotic cell via flow cytometry method. The results indicated that crocin and its liposomal form induced a sub-G1 peak in the flow cytometry histogram of treated cells, indicating apoptosis is involved in this toxicity. The results from one study demonstrated that the inhibitory activity on the in vitro growth of

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HeLa cells produced by saffron extracts (ID50¼ 2, 3 mg mL1) was mainly due to crocin (ID50 of 3 mM), whereas picrocrocin and safranal, with an ID50 of 3 and 0.8 mM, respectively, played a minor role in the cytotoxicity of saffron extracts. Microscopic studies revealed that HeLa cells treated with crocin showed vacuolated areas, size reduction, cell shrinkage, and piknotic nuclei. Changes of HeLa cells after exposure to crocin indicated the activation of program cell death pathways. In one study cervical cancer cell line (HeLa), nonsmall cell lung cancer cell line (A549), and ovarian cancer cell line (SKOV3) were treated with crocetin alone or in combination with vincristine. Data of this study has shown that crocetin has an antiproliferation effect in a concentration-dependent manner. Crocetin significantly induced cell cycle arrest through p53-dependent and -independent mechanisms accompanied with p21 (WAF1/Cip1) induction (Zhong et al., 2011).

32.9

Skin cancer

Skin cancer is one of the most common cancers in the world and is a cancer that arises from the skin. It is due to the development of abnormal cells that have the ability to invade or spread to other parts of the body. There are three major types of skin cancer: basal cell carcinoma, squamous cell carcinoma, and melanoma (Singh et al., 2014). Topical application of saffron extract (100 mg kg1 body wt.) inhibited two-stage initiation/promotion dimethylbenz[a] anthracene (DMBA)-induced skin carcinogenesis. Oral administration of saffron extract in mice after and before topical applications of DMBA inhibited the formation of skin papillomas in animals, simultaneously reduced their size and DMBAinduced skin carcinoma in mice when treated early. These authors related this effect of saffron to the induction of cellular antioxidant systems (Das et al., 2010). In addition, dose-dependent inhibitory effects of aqueous extract of saffron was found on the growth human transitional cell carcinoma and mouse nonneoplastic fibroblast cell lines (L929). It was suggested that saffron rich in carotenoids might exert its chemopreventive effects by the modulation of lipid peroxidation, antioxidants, and detoxification systems. It was demonstrated that crocetin inhibits skin tumor promotion in mice (i.e., with benzopyrene a); it has an inhibitory effect on intracellular nucleic acid and protein synthesis in malignant cells, as well as on protein kinase C, which is most likely due to its antioxidant activity (Chun et al., 2014; Magesh et al., 2006).

32.10 Prostate cancer Prostate cancer is the most common cancer among men and is a cancer that occurs in the prostate. Prostate cancer usually grows very slowly, and finding and treating it before symptoms occur may not improve men’s health or help them live longer (Schmitz-Dra¨ger et al., 2014). In a study the antiproliferative activity of saffron extract and its main constituent crocin on five different malignant and two nonmalignant prostate cancer cell lines were determined. Both saffron extract and crocin decreased cell proliferation in all malignant cell lines in a time- and concentration-dependent manner. Nonmalignant cells were not affected. The majority of cells were arrested at G0/G1 phase with a noteworthy presence of apoptotic cells based on flow cytometry profiles. The expression of Bcl-2 was strikingly down-regulated, whereas Bax was upregulated as per the western blot analysis. Analysis of caspase activity indicated a caspase-dependent pathway with involvement of caspase-9 activation, suggesting an intrinsic pathway. Dependent on these results it is suggested that both SE and crocin can inhibit cell proliferation and arrest cell cycle progression, inducing apoptosis in prostate cancer (D’Alessandro et al., 2013). Based on these findings investigators recommend that these agents potentially be used as a chemopreventive as well as a chemotherapeutic agent for prostate cancer management. Furthermore, a preclinical study demonstrated a prostate cancer cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Hence, it appears to have potential as a therapeutic agent (Samarghandian and Shabestari, 2013).

32.11 Gastric cancer Gastric cancer is a disease in which cancer cells form in the lining of the stomach. Gastric cancer remains difficult to cure, primarily because most patients present with advanced disease. A study by Bathaie et al. (2013a) showed the beneficial effect of saffron aqueous extract (SAE) on 1-methyl-3-nitro-1-nitrosoguanidine-induced gastric cancer in rats. Saffron extract administration inhibited the progression of the cancer of the gastric tissue as evidenced by the pathologic data; in fact, 20% of cancerous rats treated with higher doses of saffron extract were completely normal at the end of the experiment and there was no rat with adenoma in the saffron extract treated groups. Furthermore, the results of the flow cytometry/propidium iodide staining demonstrated that the apoptosis/proliferation ratio was augmented because

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of the SAE treatment of cancerous rats (Bathaie et al., 2013a). Thus, the investigators recommend crocin as a potential anticancer agent. The mechanism of crocin action was investigated in the gastric adenocarcinoma (AGS) cells. Crocin revealed a dose- and time-dependent cytotoxic effect against an AGS cell line. Crocin-induced apoptosis was substantiated by flow cytometry and assessing caspase activity. The improved sub-G1 population and stimulated caspases in the treated AGS cells confirmed its anticancer effect (Bathaie et al., 2013b; Williams, 2013).

32.12 Conclusion Cancer prevention by natural products could be promising in the fight against cancer and has garnered extensive consideration due to their low cost and wide safety margin. Natural compounds, particularly those obtained from plants, have been best explored for their anticancer properties and most of them have been efficient against known molecular targets of cancer. These natural products can be used in combination with modern medicine to provide better treatment of cancer. Saffron, a spice obtained from the flower of C. sativus and its constituents, appropriately act against cancer formation and exhibit selective toxicity against several cancers. Various hypotheses for the antitumor actions of saffron and its components have been proposed such as: (1) the inhibitory effect on cellular DNA and RNA synthesis, but not on protein synthesis; (2) the inhibitory effect on free radical chain reactions; (3) the metabolic conversion of naturally occurring carotenoids to retinoids; and (4) the interaction of carotenoids with topoisomerase II, an enzyme involved in cellular DNA-protein interaction. Additionally, the immunomodulatory activity on driving toward Th1 and Th2 limbs of the immune system of saffron has also been demonstrated. However, further direct evidence of the anticancer efficacy of saffron as a chemopreventive agent could be obtained from trials that use actual reduction of cancer incidence as the primary endpoint.

References Advani, P., Moreno-Aspitia, A., 2014. Current strategies for the prevention of breast cancer. Breast Cancer: (Dove Med Press) 6, 59
71. Amin, A., Hamza, A.A., Bajbouj, K., Ashraf, S.S., Daoud, S., 2011. Saffron: a potential candidate for a novel anticancer drug against hepatocellular carcinoma. Hepatology 54, 857
867. Aung, H.H., Wang, C.Z., Ni, M., 2007. Crocin from Crocus sativus possesses significant antiproliferation effects on human colorectal cancer cells. Exp. Oncol. 29, 175
180. Bajbouj, K., Schulze-Luehrmann, J., Diermeier, S., Amin, A., Schneider-Stock, R., 2012. The anticancer effect of saffron in two p53 isogenic colorectal cancer cell lines. BMC. Complement. Altern. Med. 12 (69). Available from: https://doi.org/10.1186/1472-6882-12-69. Bakshi, H., Sam, S., Rozati, R., 2010. DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri saffron in a human pancreatic cancer cell line. Asian Pac. J. Cancer Prev. 11, 675
679. Bathaie, S.Z., Hoshyar, R., Miri, H., Sadeghizadeh, M., 2013a. Anticancer effects of crocetin in both human adenocarcinoma gastric cancer cells and rat model of gastric cancer. Biochem. Cell Biol. 91, 397
403. Bathaie, S.Z., Miri, H., Mohagheghi, M.A., Mokhtari-Dizaji, M., Shahbazfar, A.A., Hasanzadeh, H., 2013b. Saffron aqueous extract inhibits the chemically-induced gastric cancer progression in the Wistar albino rat. Iran J. Basic Med. Sci. 16, 27
38. Boreddy, S.R., Srivastava, S.K., 2013. Pancreatic cancer chemoprevention by phytochemicals. Cancer Lett. 334, 86
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540. Chryssanthi, D.G., Dedes, P.G., Karamanos, N.K., Cordopatis, P., Lamari, F.N., 2011. Crocetin inhibits invasiveness of MDA-MB-231 breast cancer cells via downregulation of matrix metalloproteinases. Planta Med. 77, 146
151. Chun, K.S., Kundu, J., Kundu, J.K., Surh, Y.J., 2014. Targeting Nrf2-Keap1 signaling for chemoprevention of skin carcinogenesis with bioactive phytochemicals. Toxicol. Lett. 229, 73
84. Costantini, S., Colonna, G., Castello, G., 2014. A holistic approach to study the effects of natural antioxidants on inflammation and liver cancer. Cancer Treat. Res. 159, 311
323. D’Alessandro, A.M., Mancini, A., Lizzi, A.R., 2013. Crocus sativus stigma extract and its major constituent crocin possess significant antiproliferative properties against human prostate cancer. Nutr. Cancer 65, 930
942. Das, I., Das, S., Saha, T., 2010. Saffron suppresses oxidative stress in DMBA-induced skin carcinoma: a histopathological study. Acta Histochem. 112, 317
327. Dhar, A., Mehta, S., Dhar, G., 2009. Crocetin inhibits pancreatic cancer cell proliferation and tumor progression in a xenograft mouse model. Mol. Cancer Ther. 8, 315
323. Geromichalos, G.D., Papadopoulos, T., Sahpazidou, D., Sinakos, Z., 2014. Safranal, a Crocus sativus L. constituent suppresses the growth of K-562 cells of chronic myelogenous leukemia. In silico and in vitro study. Food Chem. Toxicol. 17, 45
50. Gutheil, W.G., Reed, G., Ray, A., Anant, S., Dhar, A., 2012. Crocetin: an agent derived from saffron for prevention and therapy for cancer. Curr. Pharm. Biotechnol. 13, 173
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82. Iovanna, J., Mallmann, M.C., Gonc¸alves, A., Turrini, O., Dagorn, J.C., 2012. Current knowledge on pancreatic cancer. Front. Oncol. 2, 6. Available from: https://doi.org/10.3389/fonc.2012.00006. Kumar, V., Bhat, Z.A., Kumar, D., Khan, N.A., Shah, M.Y., 2011. Pharmacological profile of Crocus sativus – a comprehensive review. Pharmacologyonline 3, 799
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8. Samarghandian, S., Borji, A., 2014. Anticarcinogenic effect of saffron (Crocus sativus L.) and its ingredients. Phcog. Res. 6, 99
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183. Samarghandian, S., Boskabady, M.H., Davoodi, S., 2010. Use of in vitro assays to assess the potential antiproliferative and cytotoxic effects of saffron (Crocus sativus L.) in human lung cancer cell line. Phcog Mag. 6, 309
314. Samarghandian, S., Tavakkol-Afshari, J., Davoodi, S., 2011. Suppression of pulmonary tumor promotion and induction of apoptosis by Crocus sativus L. extraction. Appl. Biochem. Biotechnol. 164, 238
247. Samarghandian, S., Borji, A., Farahmand, S.K., Afshari, R., Davoodi, S., 2013. Crocus sativus L. (saffron) stigma aqueous extract induces apoptosis in alveolar human lung cancer cells through caspase-dependent pathways activation. Biomed. Res. Int. 417928. Available from: https://doi.org/ 10.1155/2013/417928. Sasieni, P., 2006. Chemoprevention of cervical cancer. Best Pract. Res. Clin. Obstet. Gynaecol. 20, 295
305. Schmitz-Dra¨ger, B.J., Scho¨ffski, O., Marberger, M., Sahin, S., Schmid, H.P., 2014. Risk adapted chemoprevention for prostate cancer: an option? Recent Results Cancer Res. 202, 79
91. Singh, A.A., 2009. India can do more for breast and cervical cancer control. Asian Pac. J. Cancer Prev. 10, 527
530. Singh, M., Suman, S., Shukla, Y., 2014. New enlightenment of skin cancer chemoprevention through phytochemicals: in vitro and in vivo studies and the underlying mechanisms. Biomed. Res. Int. 243452. Available from: https://doi.org/10.1155/2014/243452. Sun, Y., Xu, H.J., Zhao, Y.X., 2013. Crocin exhibits antitumor effects on human leukemia HL-60 cells in vitro and in vivo. Evid. Based Complement. Alternat. Med. 690164. Available from: https://doi.org/10.1155/2013/690164. Vineis, P., Wild, C.P., 2014. Global cancer patterns: causes and prevention. Lancet 383, 549
557. Williams, C.D., 2013. Antioxidants and prevention of gastrointestinal cancers. Curr. Opin. Gastroenterol. 29, 195
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1536.

Chapter 33

Available saffron formulations and product patents Seyed Ahmad Mohajeri1,2, Narges Hedayati3 and Mehri Bemani-Naeini4 1

Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran,

2

Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran,

3

Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 4Nanotechnology Research Center,

Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 33.1 Introduction 33.2 Skin care products 33.2.1 Creams 33.2.2 Masks 33.3 Health care products 33.3.1 Toothpaste 33.3.2 Drugs for kidney health 33.3.3 Compositions containing enriched natural crocin and/or crocetin 33.3.4 Hair conditioner 33.4 Therapeutic products 33.4.1 Chinese medicine for treatment of angiitis 33.4.2 Krocina tablet 33.4.3 Topical spray 33.4.4 Arthritis and rheumatism liquid patch 33.4.5 Ointment for treating dermatitis 33.4.6 Antiacne ointment 33.4.7 Topical spray for treating skin scars 33.4.8 Oral compositions 33.4.9 Tibetan nighttime medicine 33.4.10 Antimicrobial composition 33.4.11 Chinese medicinal composition 33.4.12 Gout medicine 33.4.13 Prepregnancy fetus protection pills 33.4.14 Cold and flu symptomatic relief composition 33.4.15 Herbal composition for treating diabetes 33.4.16 Multiglycosides saffron tablets 33.4.17 Externally applied Chinese medicine 33.4.18 Satiation agent for the treatment of obesity 33.4.19 Medicine for treating chronic obstructive pulmonary disease 33.4.20 Topical treatment for rheumatic arthritis 33.4.21 Chinese medicine for treatment and prevention of rheumatic arthritis

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Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00034-4 © 2020 Elsevier Inc. All rights reserved.

33.4.22 Saffron-based compositions for treating of duodenal bulbar ulcer or inflammation 33.4.23 Traditional Chinese medicine for treating gastric ulcer 33.4.24 Topical treatment for breast cancer 33.4.25 Chinese medicine for treating brain apoplexy 33.4.26 Saffrotin capsule 33.4.27 Composition for the treatment and prevention of degenerative eye disorders 33.4.28 Medicine for treating prostatic hyperplasia 33.4.29 Chinese medicine for treatment of gynecologic diseases 33.4.30 Traditional Chinese composition for treating dysmenorrhea 33.4.31 Composition for treating endocrine dysfunction 33.4.32 Chinese formulation for treating premature ovarian failure 33.4.33 Traditional Chinese medicine for treating rheumatic heart disease 33.4.34 Chinese medicine for treating cataracts 33.4.35 Traditional Chinese medicine capable of treating cervical spondylosis 33.4.36 Traditional Chinese medicine preparation for treating qi stagnation and blood stasis 33.4.37 Chinese medicine for treating osteoproliferation and herniated disk 33.4.38 Traditional Chinese medicine for treating blocking antibody deficiency in recurrent spontaneous abortion 33.4.39 Herbal medicine formula for treating nasopharyngitis 33.4.40 Chinese medicine for treating lung tumor 33.4.41 Medicine for treating damp-heat stagnation (abdominal mass)

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33.4.42 Traditional Chinese medicine composition for treating ascites due to cirrhosis 33.4.43 Traditional Chinese medicine for treating nonulcer dyspepsia 33.4.44 Chinese medicine for treating peptic ulcer 33.4.45 Medicine for treating ankylosing spondylitis 33.4.46 Externally-applied wet tissue for treating measles 33.4.47 Treatment of herpes zoster 33.4.48 Chinese composition for treating septic shock

33.1

503 503 503 503 503 503 504

33.4.49 Drug for treating bone injury 33.4.50 Traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases 33.4.51 Medicine for treating eyelid eczema 33.5 Food products 33.5.1 Vegetable drinks 33.5.2 Healthy drink prepared from saffron pollen 33.6 Conclusion References

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Introduction

Saffron (Crocus sativus L., Iridaceae), the most expensive natural product, is also known as “red gold” and has been widely used in the cosmetic and food industries. It also has numerous proven benefits such as antiinflammation, antitumor, and anticonvulsant functions in cancer; for the prevention of cardiovascular disease; use in phototherapy; and in the treatment of neurodegenerative diseases. In addition, saffron has the ability for scavenging free radicals, and is also involved in the improvement of depression and memory loss (Hosseini et al., 2018). Among the components of saffron that are involved in determining saffron quality, crocin (gives saffron its color), picrocrocin (gives saffron its taste), and safranal (gives saffron its aroma) are the key pharmacologically active substances (Aliakbarzadeh et al., 2016). There is a growing body of patents utilizing saffron and its components, alone or in combination with the appropriate raw materials, in several therapeutic or pharmaceutical fields. Nonetheless, the clinical use of these compounds are limited due to the lack of adequate clinical trial data. This lack has required that more attention be devoted to translating the use of saffron from its theoretical use to its practical use (from “bench to bedside”), especially as herbal compounds grow in demand on a global scale. This chapter covers the findings and product patents that emphasize the therapeutic and cosmetic properties of saffron and its derivatives. These properties qualify the outstanding importance of saffron, crocin, crocetin, picrocrocin, campherol, and safranal on human health and diseases.

33.2

Skin care products

33.2.1 Creams 33.2.1.1 Bath cream Bath cream was prepared from saffron by Cheng et al. and is comprised of citron fruit water, surfactant, glycerol monooleate, dry saffron, plant extract, orange flower oil, lactic acid, glycerol, benzyl alcohol, and deionized water. The saffron bath cream has beneficial effects on the body, including refreshing and whitening the skin, accelerating blood circulation, and resisting the effects of aging (Cheng et al., 2015).

33.2.1.2 Face cream Face cream is a saffron product that beautifies skin and makes it healthy. The saffron in face cream has an important role in nourishing the blood and reducing hyperpigmentation. After continuous use of this cream twice daily, in both the morning and evening, the skin obtains a youthful appearance (Yaocheng, 2006).

33.2.1.3 Hand cream Herbal hand cream is able to protect the skin on the hands from becoming too dry. Traditional creams are less effective than the herbal creams because of the fat content and lack of nourishing ingredients. Presently, this hand cream solves these problems and the saffron extract in this cream possesses analgesic and antiinflammatory properties. The saffron in hand cream also prevents the reddening of skin and adds shine (Baoquan, 2016).

33.2.1.4 Biological cosmetic cream Biological cosmetic cream is based on the traditional Chinese medicine, containing bee ova, grub, honey, saffron, and wine. This cosmetic cream is applied for removing spots, cyasma, butterfly spots, and acne (Jitong et al., 2009).

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33.2.1.5 Freckle-whitening cream This whitening cream is comprised of raw materials including ginseng, honeysuckle, saffron, licorice, chuan shao, angelica, glycerol, polygonum, white peony root (Paeonia lactiflora), melothria, white monkshood (Aconitum napellus), atractylodes, radix, fritillaria, ebony (Diospyros ebenum), epimedium, cyperus, and motherwort (Nepeta cataria). This product is effective for elimination of melanin and shrinkage of skin pores. Also, its use helps to resist aging, boost metabolism, activate blood circulation, remove stasis, and detoxify the skin from mercury, lead, and toxin. Frecklewhitening cream can also repair black spots, cyasma, chloasma, rebounding freckles, sunburn, black skin, yellow skin, and coarse skin (Li, 2009).

33.2.1.6 Aloe whitening cream Saffron, with its strong physiological activity, is one of the main ingredients of this whitening cream. It has an important role in increasing blood circulation and cooling blood detoxification. Aloe whitening cream contains saffron that brightens the skin and possess mild moisturizing benefits (Zhenbiao, 2015).

33.2.1.7 Fu Yang repair cream This repair cream contains saffron, collagen jelly, red ginseng, celery, vitamin C and E, etc. The main advantages of this invention are that it improves wrinkling and fine lines in the skin around the eyes, reduces puffiness, dark circles, and bags under the eyes, and repairs damaged skin. Every night before sleep, Fu Yang repair cream should be used in the area around the eye according to the product directions (Yang, 2016).

33.2.1.8 Liquid cream This cosmetic product is prepared from the following raw materials: saffron crocus, muskone, sage (Salvia miltiorrhiza) powder, pearl powder, radix paeoniae alba, aloe, and nopal (Opuntia sp.). Liquid cream shows significant effects on pigmentation disorder and acne. Moreover, the skin becomes white, tender and rich in elasticity after continuous use of the liquid cream (Yuhong, 2011).

33.2.1.9 Fair-complexion face powder This powder, which was invented by a Chinese group, is appropriate for hydrating, moisturizing, whitening, brightening, fading acne, and smoothing skin. Indeed, fair-complexion face powder is produced without lead, mercury, arsenic, and other harmful ingredients, which appears to prevent many serious side effects of extended use (Yi, 2012).

33.2.2 Masks 33.2.2.1 Rose mask Rose mask is comprised of two main components: dry roses and dry saffron. Alcohol and fragrance is excluded during the formulation of rose mask, which makes the product more safe to use. The benefits of this mask are whitening of skin, elimination of freckles, and improved skin elasticity. Another advantage of this product is that it is available at a low cost (Hao and Wei, 2013).

33.2.2.2 Mask for treating common acne This acne mask is comprised of 80% saffron extract and 6% saffron essential oil and is utilized to effectively treat common acne and sores such as acne vulgaris. The presence of saffron in the mask activates blood circulation and causes reduced swelling as well as the evacuation of pus (Chen et al., 2015).

33.2.2.3 Oil-induced acne herbal soap This herbal acne-removing soap is comprised of pure natural plants such as ginseng, saffron, angelica, cnidium, and peach (Prunus persica) bitter parts. Long term use of this soap, based on a claim from the inventors, can improve facial

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wrinkles, fine lines, capillary circulation, metabolism, and nourish damaged skin. Moreover, the soap removes dull, yellowed, and couperose skin in addition to its acne benefits (Zhen, 2015).

33.2.2.4 Attapulgite-saffron suntan lotion This suntan lotion contains saffron and possesses a combination of physical and chemical sun-screening properties that are able to reflect ultraviolet light. Attapulgite-saffron suntan lotion forms a protective layer to protect the skin from ultraviolet rays. The saffron ingredient accelerates blood circulation, which causes the skin gets plenty of oxygen, water and nutrients (Wenling et al., 2009).

33.3

Health care products

33.3.1 Toothpaste This herbal toothpaste contains Tibetan worm grass (Ophiocordyceps sinensis), fritillaria, and saffron crocus. This herbal toothpaste acts as a medicine to help improve gum problems, improve health of the periodontium, whiten the teeth, and nourish the gingiva (Russian, 2015).

33.3.2 Drugs for kidney health Drugs for kidney health that contain saffron and other pharmaceutical raw materials were prepared in capsule and pill form by a Chinese group. Excessive use of this drug causes the enhancement of sexual desire, sexual energy, and stronger ejaculation. In addition, the use of kidney drugs causes an increase in semen frequency and micturition urgency without any adverse side effects (Yucong and Kunming, 2010).

33.3.3 Compositions containing enriched natural crocin and/or crocetin Compositions that contain mainly enriched or purified natural crocin or crocetin are used alone or in combination with other health phytochemicals in nutraceutical or therapeutic treatments. This healthy composition can be utilized as a functional food, drink or dietary supplement, or administered (orally, etc.) in therapeutic dosages in humans to prevent or treat cancers and other diseases (Gao, 2016).

33.3.4 Hair conditioner This hair conditioner is comprised of saffron crocus, Polygonum multiflorum, licorice, honey suckle (Lonisera sp.), lecithin, casein, amino acid, radix stemonae, methyl parahydroxybenzoats cassia twig (Cinnamomum cassia), essence, and deionized water. This saffron-based hair conditioner has some advantages such as simple preparation, and low cost, which presents no harm to the hair (Yan, 2013).

33.4

Therapeutic products

33.4.1 Chinese medicine for treatment of angiitis The main ingredients of this medicine are saffron, white pepper, cow’s periost, pearl powder, tortoise plastron glue, jujube, and Chinese angelica [Angelica sinensis (female ginseng)] root. This pure Chinese medicine is used for curing angiitis without surgical repair of vasculitis and deep vein occlusion. This Chinese medicine has shown clinical efficiency up to 98% (Zuohua, 2000).

33.4.2 Krocina tablet Krocina tablet, which was invented by an Iranian group, contains crocin, a carotenoid chemical compound and the main active constituent of saffron. This tablet is prescribed for depression, Alzheimer’s disease, sexual dysfunction, metabolic

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syndrome, diabetes, burning mouth syndrome, and age-related macular degeneration. Moreover, this product can prevent several cancers (Mohajeri et al., 2014).

33.4.3 Topical spray Topical spray containing saffron is used for treatment of rheumatoid bone disease. This spray is absorbed through the skin and acts directly on the affected area without any side effect or pain. In comparison with other existing therapies, this product possesses a high success rate and is convenient to use (Hai, 2014a).

33.4.4 Arthritis and rheumatism liquid patch This liquid patch is comprised of herbal extracts including saffron, panax, ginger (Zingiber officinale), achyranthes mint, plasticizers, and a film-forming material. The liquid patch is applied for treating arthritis and rheumatism. This product successfully resolved different issues seen in similar patches, such as the permeability problems involved in skin stimulation of the plaster patch. Other advantages of this liquid patch are that it is low in cost, convenient to carry and use, and environmentally friendly (Ziqiang et al., 2016).

33.4.5 Ointment for treating dermatitis This ointment, produced from saffron stigma, is appropriate for treating dermatitis. It appears to show promising therapeutic effects without any irritation, and therefore has little adverse side effects on the human body (Jian, 2017).

33.4.6 Antiacne ointment Antiacne ointment, which is formulated with herbal medicine, is effective to treat acne conditions, especially acne vulgaris common in adolescents (Xiaorui, 2011).

33.4.7 Topical spray for treating skin scars This herbal spray, which is used for curing skin cicatrix, is comprised of eight Chinese medicinal components, including but not limited to ginseng, Chinese angelica root, saffron, and borneol. These ingredients are combined proportionally, immersed in liquor and sealed to create the topical spray. This product is sprayed 6
8 times per day directly on the patient’s skin scar for a duration of 3
6 months to eliminate the skin cicatrix. The advantages of this spray are that it is available for a low cost, is simple to prepare, highly effective for removing skin scars, has no toxic side effects, and no adverse reactions (Xiuyun, 2007).

33.4.8 Oral compositions Oral compositions are prepared from mint essential oil, Curcuma longa derivatives, Olea europea derivatives, and possibly one or more of the following: N-acetylcysteine, glutathione, ubidecarenone, lactoferrin, carotenoid, polyphenol, vitamin C, vitamin E, extract or derivative of St. John’s wort (Hypericum perforatum), kava kava (Piper methysticum), saffron, valerian, passion flower, chamomile, and griffonia. This preparation is used for the prevention of inflammatory colon disorders. The release of this composition in the colon can solve problems of reflux and esophagitis through the properties of the mint oil. Moreover, curcumin adds antiinflammatory action to the composition. The mixture of components obviously decreases the time of response (from 6
7 to 4
5 days), with an efficacy rate ranging between 85% and 95% (Tramonti, 2009).

33.4.9 Tibetan nighttime medicine This medicine contains saffron, which is prepared in various form such as pills, powders, capsules, soft capsules, and granules for the treatment of diabetes. This product can efficiently reduce blood sugar and serum triglycerides.

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This drug boasts advantages such as quick absorption, high therapeutic effect, easy consumption, and a lack of toxic side effects (Wei, 2009).

33.4.10 Antimicrobial composition This antimicrobial product includes natural ingredients such as Curcuma longa, C. sativus (saffron), Alkanna tinctoria (dyer’s alkanet) and Hydrastis canadensis (golden seal) used for disinfecting the skin. This product also has antiinflammatory properties that can treat skin inflammation and bacterial infections such as acne, pseudofolliculitis, localized redness, and localized odor (Jampani et al., 2013).

33.4.11 Chinese medicinal composition The raw materials of the Chinese medicinal composition are saffron, zaopi, zao ren, wolfberry (Lycium barbarum), arsenic, indigo gray, and borneol. This product is effective to treat acute and chronic oral diseases. It is utilized to improve inflammation, pain, bleeding, swelling, tissue regeneration effect, convergence wounds, stasis, acute, and chronic apical periodontitis, gingivitis, halitosis, pulpitis, and abscesses. Beneficially, it has significant therapeutic properties, works quickly, and has a 100% efficiency rate and a 95% cure rate (Xiuzhen, 2002).

33.4.12 Gout medicine This gout medicine includes saffron as one of the natural Chinese medicinal herbs that helps to treat gout. Gout is caused by increased uric acid blood concentration due to purine metabolism disorder (Wenju, 2016).

33.4.13 Prepregnancy fetus protection pills These pills are formulated by using components such as large head atractylodes (Atractylodes macrocephala) rhizome, saffron, eucommia [Eucommia ulmoides (Eucommiaceae)], Chinese eaglewood (Aquilaria sinensis (Thymelaeaceae)], baikal skullcap root [Scutellaria baicalensis (Lamiaceae)], dipsacus root, malaytea scurfpea [Psoralea corylifolia (Fabaceae)] fruit, selenium element, and an antiabortifacient. The pills work by assisting with reproduction, regulating the sex of the fetus, and preventing miscarriage. The fetus prevention pills also support healthy energy levels, spleen tonification, liver and kidney regulation and tonification, bone strengthening, and fetus fixing. The pills are principally suitable to be used before pregnancy for selecting the fetus sex. A small dose of this product is very potent in the promotion of fetal development. Notably, while taking these pills, the health of pregnant women is not affected (Jing, 2012).

33.4.14 Cold and flu symptomatic relief composition The formulation of this composition includes extracts of black pepper, cumin (Cuminum cyminum), ginger, turmeric [C. longa (Zingiberaceae)], cinnamon (Cinnamomum verum), rose hip, and saffron, and is applied to alleviate suffering from the common cold or flu. In this product, saffron is included because of its antioxidant and anticarcinogenic properties (Gopinathan, 2011).

33.4.15 Herbal composition for treating diabetes This herbal composition is a mixture of medicinal herbs including dried saffron packaged in a teabag. It is used to enhance insulin excretion in the treatment of type I diabetes (Mashat Ba and Ihab, 2015).

33.4.16 Multiglycosides saffron tablets In multiglycosides tablets that can be used for treatment and prevention of cardiovascular disease, saffron promotes blood circulation. This tablet can effectively inhibit platelet aggregation, which induces thrombus formation, and improves heart coronary blood flow (Fengyun, 2003).

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33.4.17 Externally applied Chinese medicine This product is made of saffron, safflower, Ligusticum wallichii, bitter bamboo leaf, belamcanda rhizome, and calcarea lime and is externally applied for treating ulcerative stomatitis. This drug can reduce healing time and provide better pain relief for those suffering from this disease (Fengyun, 2006).

33.4.18 Satiation agent for the treatment of obesity The function of the satiation agent is to treat obesity, control body weight, and regulate the intake of calories consumed daily. The product is comprised of saffron and its active ingredients: safranal, picrocrocin and crocin (Bourges, 2010).

33.4.19 Medicine for treating chronic obstructive pulmonary disease This formulation is prepared from raw materials such as saffron, codonopsis, Chinese yam (Dioscorea polystachya), asters, tinglizi (Descurainia sophia), acanthopanax [Kalopanax septemlobus (Araliaceae)], epimedium, maodongqing [Ilex pubescens (Aquifoliaceae)], gold boiling grass, nepeta, angelica, etc. The formulation is used for curing asthma, cough symptoms, and phlegm as well as for boosting the bodily immunity, promoting regeneration of respiratory mucosa of damaged epithelium, development of decreased pulmonary functions, and controlling the progress of disease (Feng and Wenkui, 2014).

33.4.20 Topical treatment for rheumatic arthritis This drug is produced for treating rheumatic arthritis disease. It is comprised of these materials: saffron, krait/agkistrodon, centipede, black-striped snake, salvia root, ephedra, large gentian (Gentiana lutea) root, notoginseng (Panax notoginseng), etc. The topical application is useful for suppressing inflammation, frozen shoulder, cervical pain, statis, numbness of the extremities, rheumatic arthritis, sciatica, and scapulohumeral periarthritis. It is also useful in boosting blood circulation. The advantages of using this product is its high therapeutic rate and that it does not cause any skin irritation or toxic side effects (Shuren, 2002).

33.4.21 Chinese medicine for treatment and prevention of rheumatic arthritis This medicine is made of cassia twig, longhairy antenoron herb [Lysimachia christiniae (Primulaceae)], semen strychni (Strychnos nux-vomica), divaricate saposhnikovia [Saposhnikovia divaricate (Apiaceae)] root, processed manis pentadactyla, common andrographis herb, cortex acanthopanacis, saffron, notopterygium [Notopterygium incisum (Apiaceae)] root, burdock (Arctium tomentosum), fructus forsythiae (Forsythia 3 intermedia), pubescent angelica (Angelica pubescens) root, etc. This Chinese medicinal formulation is capable of preventing and treating the symptoms of rheumatic arthritis (Huile, 2016).

33.4.22 Saffron-based compositions for treating of duodenal bulbar ulcer or inflammation This Chinese medicinal formulation is prepared from the following raw materials: licorice, safflower, red dates, honey, and saffron. This saffron-based composition is effective in preventing duodenal ulcer or inflammation; fortunately, it is highly effective and is available at a low cost (Kong et al., 2007).

33.4.23 Traditional Chinese medicine for treating gastric ulcer The present invention has remarkable treatment effects on gastric ulcers. It is comprised of raw materials commonly used in Chinese medicines such as: nard (Nardostachys jatamansi), saffron, bai caoshuang, mango nuclear, malan, beans, bamboo pepper, aeschynomene, hungry leeches (Chtonobdella limbata), kowloon root (Ficus microcarpa), good vine, turnip (Brassica rapa subsp. rapa), onion skin, and revive rattan grass. The advantages of this formulation is its effectiveness in treating epigastric pain, gastric and duodenal ulcers, improvement of gastric motility, recovery

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of normal stomach metabolism, and quickness of treatment response with no toxic side effects (Guorong and Zejin, 2014).

33.4.24 Topical treatment for breast cancer This topical-use treatment for breast cancer is prepared from 19 Chinese-medicinal materials including but not limited to saffron, astragalus root, forsythia fruit, burreed tuber (Sparganium stoloniferum), zedoary (Curcuma zedoaria), and curcuma root (C. longa). The treatment is also appropriate for curing superficial skin tumors. The topical treatment is highly effective with a success rate of up to 92.3%. This product results in less skin irritation than the alternatives and has no toxic side effects (Zhenfeng, 2000).

33.4.25 Chinese medicine for treating brain apoplexy This medicine for treating cerebroplegia, cardiovascular disease, and cerebrovascular diseases is prepared from medicinal ingredients such as saffron, baizhu (A. macrocephala), leech peony shichangpu, salvia chuanxiong (Salvia chuanxiensis), achyranthes large bezoar, chicken blood (Teng Teng), peach angelica, safflower guizhi, shanjia beads astragalus wind, silkworm scorpion, hematoxylin motherwort, jatamansi sunburn strychnos powder, etc. (Jixu, 2000).

33.4.26 Saffrotin capsule Saffrotin, an Iranian medicine available in capsule form, is comprised of crocin, an active component of saffron. This product was invented for the reduction of Alzheimer’s disease symptoms. These capsules can decrease nervous system apoptosis, production of reactive oxygen species, and oxidative stress. Saffrotin also prevents death and nervous cell atrophy and increases serotonin activity (Mafi, 2014).

33.4.27 Composition for the treatment and prevention of degenerative eye disorders This oral treatment includes saffron and curcumin for the prevention and treatment of degenerative eye disorders. The concomitant use of saffron and curcumin show a synergistic effect and produce greater activity than a composition comprising only curcumin or saffron alone (Bisti et al., 2017).

33.4.28 Medicine for treating prostatic hyperplasia This medicine is used in the treatment of prostatic hyperplasia caused by kidney qi deficiency. It is comprised of components such as: radix astragali, arillus longan (Euphoria longan), rhizoma A. macrocephala, fructus corni, cortex moutan, S. miltiorrhiza, Acorus gramineus, radix bupleuri (Bupleurum chinense and Bupleurum scorzonerifolium), Pyrrosia lingua, combined spicebush (Lindera aggeregata) root, mulberry fruit (Morus nigra), wolfberry fruit, Glechoma longituba, spica prunellae (Prunella vulgaris), white peony root, cassia twig from szechwan, C. sativus, Hedyotis diffusa, medulla junci (Juncus effusus), cinnamon, carapax testudinis, and rhizoma alismatis (Alisma orientalis). The advantages of this traditional Chinese medicinal decoction includes: low cost, availability, lack of toxic side effects, ability to use it for treating nephrotic syndromes and concurrent kidney nourishing, yang warming, eliminating stagnation, tonifying qi, removing stasis, alleviating water retention, and treating stranguria. It should be taken daily in both the morning and evening for 20 days with intervals of 3 days between each treatment course (Shuhua, 2012).

33.4.29 Chinese medicine for treatment of gynecologic diseases The medicine for treating gynecologic diseases was prepared from the following raw materials: olive oil, C. sativus flower, P. multiflorum, Poria cocos, A. sinensis, Cyperus rotundus, Spatholobus suberectus stem, Caesalpinia sappan, and herba asari (Asarum heterotropoides and/or Asarum sieboldii). The advantages of this product are its simple preparation method, low cost, convenience to use, significant therapeutic effects, and pain-free treatment (Hai, 2014b).

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33.4.30 Traditional Chinese composition for treating dysmenorrhea This herbal composition consists of the following materials: salvia, vinegar fumarate, saffron, white wine shao, wine angelica, march, motherwort, cyperus, toosendan (Melia azedarach), and bupleurumand honey-roasted cumin licorice. This drug has effects on promoting blood circulation, treating dysmenorrhea, invigorating qi, nourishing blood, and relieving depression of the liver-qi. It also shows definite efficacy, quick onset of action, and few side effects (Mei, 2013).

33.4.31 Composition for treating endocrine dysfunction This traditional Chinese medicine is used for treatment of endocrine dysfunction, and is comprised of mixing raw materials of fineleaf schizonepeta herbs (Nepeta tenuifolia), moxa liquoric (Glycyrrhiza glabra) roots, white muscardine silkworm, notopterygium (N. incisum) roots, balloon flowers (Platycodon grandiflorus), Chinese thorowax roots (B. chinense and B. scorzonerifolium), Cremastra appendiculata, Lycopus lucidus, oriental wormwood (Artemisia annua), Geranium strictipes, roselle (Hibiscus sabdariffa), Leonurus macranthus, mioga ginger (Zingiber mioga), C. sativus, herba hyperici japonici (Hypericum japonicum), P. notoginseng, heartleaf houttuynia herbs (Houttuynia cordata), Chinese angelica, bighead atractylodes rhizome (A. macrocephala), white peony roots, cinnamon, etc. Being simple in manufacturing and having a high absorption effect, long duration of efficacy, significant curative effect, no adverse side effects, and low cost of production are some of the benefits of this medicine (Yueqin, 2015).

33.4.32 Chinese formulation for treating premature ovarian failure This invention belongs to the technical field of traditional Chinese medicine, and is the formula used for treating premature ovarian failure. This formulation is prepared from the following medicinal raw materials: Boschniakia rossica, Lethariella zahlbruckneria, gorse (Ulex europaeus), sparrow, licorice, lanatechead saussurea herb (Saussurea costus) with flower, polyalthia root (Polyalthia longifolia), Muraenesox cinereus, Polygonatum kingianum, cucubalus root (Silene cucubalus), parts of C. sativus, etc. This product has positive effects on invigorating the spleen and stomach, nourishing yin, liver and kidney, replenishing qi, increasing body fluid production, and improving the bodily immunity, and can remarkably treat premature ovarian failure (Ping et al., 2015).

33.4.33 Traditional Chinese medicine for treating rheumatic heart disease A Chinese group produced an oral formulation for treatment of rheumatic heart disease, which is composed of radix glycyrrhizae preparata, dried tangerine peel (Citrus reticulata), cassia twigs, Codonopsis pilosula, C. sativus, radix paeoniae rubra (Paeonia officinalis), Astragalus membranaceus, radix aconiti lateralis preparata, rhizoma atractylodis, cortex acanthopanacis, and Coix lachrymal. This traditional Chinese medicinal product promotes blood circulation in the treatment of rheumatic heart disease (Jianli, 2015).

33.4.34 Chinese medicine for treating cataracts This composition of butterfly bush flower (Buddleja davidii), selfheal (P. vulgaris), black soybean flower, semen cassia, fructus lycii, Scrophularia ningpoensis, radix curcumae, ternate grape fern herb, hollow azurite, Marsh equisetum, C. sativus, boa gall, littletooth hydrangeavine root, and Cardamine lyrata is effective for treating cataracts. This product should be consumed twice daily for 30 days per course of treatment. It is highly safe to use, has a low recurrence rate after recovery, boasts a high cure rate, and has no toxic effects (Hefang, 2015b).

33.4.35 Traditional Chinese medicine capable of treating cervical spondylosis The main components of this traditional Chinese medicine preparation, which is used for treating cervical spondylosis, are A. sinensis, the rhizome of chuanxiong, radices sileris peach kernel, C. pilosula, A. membranaceus, pawpaw (Asimina triloba), caulis spatholobi (S. suberectus), ground beetle, radix clematidis (Clematis chinensis), root of bidentate achyranthes, parasitic loranthus (Loranthus sp.), notopterygium root, Curcumae radix, C. sativus, and Asarum known as wild ginger. Benefits of using this product are: low cost of treatment, no toxic side effects, easy to relapse

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after healing, major reduction of the suffering, 100% effectiveness, and a high cure rate. Treatment consists of two times per day for 30 days (Fen, 2014).

33.4.36 Traditional Chinese medicine preparation for treating qi stagnation and blood stasis Raw materials of this Chinese medicine are mulberry, nothopanax, Salvia evansiana, Salvia bowleyana, Salvia chinensis, C. sativus, sinkiang corydalis (Corydalis nobilis) tuber, Callosciurus erythraeus powder, all grass of sinuate tickclover, folium apocyni veneti (Apocynum venetum), Jasminum grandiflorum, rhizoma cyperi, fingered citron (Citrus medica var. sarcodactylis), and licorice. This product is used for treating qi stagnation and blood stasis amenorrhea, and is able to control vital energy, soothe the liver, promote blood circulation, and eliminate stagnation. The preparation should be taken three times a day (Li, 2014).

33.4.37 Chinese medicine for treating osteoproliferation and herniated disk This Chinese medicine, which is used for treating osteoproliferation and herniated disks, is comprised of wine-fried Chinese angelica, divaricate saposhnikovia root, C. sativus, vinegar-processed pangolin scales, dragon’s blood (Croton lechleri), root of red-rooted salvia (S. miltiorrhiza), wine-fried rhubarb (Rheum rhabarbarum), vinegar-processed corydalis (Corydalis yanhusuo) tuber, vinegar-processed frankincense (Boswellia sacra), myrrh (Commiphora myrrha), medicinal cyathula root (Cyathula officinalis), scalded drynaria rhizome (Drynaria roosii), etc. This product has the advantages of having no side effects, low toxicity, and being highly effective to reduce symptoms with a 97% success rate. It is suggested to use this medicine three times daily for 30 days; usually 1
6 courses are enough to achieve expected results (Shichang et al., 2012).

33.4.38 Traditional Chinese medicine for treating blocking antibody deficiency in recurrent spontaneous abortion This product is used for treating blocking antibody deficiency in recurrent spontaneous abortion (RSA). The deficiency of blocking antibodies is a major source of recurrent feticides. Due to the lack of such antibodies, which protect normal pregnant mothers against miscarriage, the bodies of pregnant women may abort their fetus. This medicine is made of the following traditional Chinese medicinal materials: human placenta, herba epimedii (Epimedium brevicornum), C. sativus, Morinda officinalis, radix astragali (Astragalus propinquus), ginseng, semen cuscutae (Cuscuta chinensis), stirfried radix paeoniae alba (P. lactiflora), stir-fried rhizoma A. macrocephala, licorice, male silkworm moth, rhizoma polygonati, corn cervi pantotrichum, Cynomorium songaricum, herba cistanche, gecko, walnut, etc. (Enxue, 2012).

33.4.39 Herbal medicine formula for treating nasopharyngitis Herbal medicine for the treatment of nasopharyngitis is made of raw materials including Scaphium scaphigerum, Platycodon grandiflorum, radix scrophulariae, herba rabdosiae [Isodon rubescens (Lamiaceae)], dried radix rehmanniae (Rehmannia glutinosa), Rabdosia rubescens, Dendrobium nobile, American ginseng (Panax quinquefolius), mint, olive, C. sativus, radix ophiopogonis, isatis root (Isatis tictoria), etc. This formula has advantages of producing significant treatment results without any side effects (Xiaoqing et al., 2014).

33.4.40 Chinese medicine for treating lung tumor This invention is comprised of C. sativus, Cordyceps sinensis, radix scrophulariae (Scrophularia buergriana), radix ophiopogonis, licorice, honeysuckle, rose root, rhizoma Atractylodis, and Ganoderma lucidum. The advantages of this medicine is its rapid absorption, high efficacy, immediate pain relief, safety, convenience of use, suitability for wide application, and lack of side effects including irritation (Yulian and Chaobin, 2014).

33.4.41 Medicine for treating damp-heat stagnation (abdominal mass) The present invention for treating depression syndrome type heat Zhengjia disease medicine is comprised of the following ingredients: berberine, tieshu (Dracaena fragrans), cicada insects, palmetto sub, saffron, bell capillaris, verbena,

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ganoderma (G. lucidum), licorice, etc. It should be used orally three times a day—morning, afternoon, and evening (Sun, 2014).

33.4.42 Traditional Chinese medicine composition for treating ascites due to cirrhosis This invention utilizes a traditional Chinese medicine formula for treating ascites due to cirrhosis. The composition is prepared from herbal medicines such as Chinese lobelia (Lobelia chinensis), wrightia pubescens (Wrightia pubescens), Cynanchum paniculatum, semen cassia, Phyllodium pulchellum, Sculellaria barbata, rhizoma cyperi, L. wallichii, C. sativus, S. chinensis, Curcuma aromatica, etc. The invention treats cirrhosis-induced ascites by boosting qi circulation, clearing liver heat, alleviating depression, and detoxifying and promoting blood circulation. This results in removing blood stasis, inducing diuresis to relieve edema, dispelling wind, removing dampness, nourishing blood and liver, replenishing qi to invigorate spleen, modulating middle energizer, and tonifying qi. This product has the advantages of having great pharmacological function, exacting efficacy, quick effect, and compatibility with other drugs (Guohua, 2015).

33.4.43 Traditional Chinese medicine for treating nonulcer dyspepsia This medicine aims to provide a therapeutic purpose for treating nonulcer dyspepsia. This traditional Chinese medicine composition contains the following materials: herba epimedii, common curculigo rhizome (Curculigo orchioides), Wolfiporia extensa, barbary wolfberry fruit, A. sinensis, radix paeoniae alba, licorice, C. pilosula, R. glutinosa, C. sativus, rhizoma cyperi, turmeric root tuber (C. longa), etc. This product plays a role in replenishing bone marrow and promoting blood circulation, which leads to removing stasis, improving metabolism and gastroenteric functions, enhancing the secretion of immune factors, promoting body immunity, naturally promoting health qi, strengthening the body, and prolonging life span (Shulin, 2015).

33.4.44 Chinese medicine for treating peptic ulcer The Chinese medicine preparation for treating peptic ulcers is comprised of the following materials: radix bupleuri, Pistacia lentiscus, medicated leaven, C. sativus, lignum sappan (C. sappan), Polygonatum sibiricum, Malus baccata, fritillary, radix glycyrrhizae, rhizoma corydalis, etc. The composition has the advantages of purity, low toxicity, no side effects, good therapeutic effect, short course of treatment, high cure rate for peptic ulcer, and low-cost of raw materials (Dong et al., 2015).

33.4.45 Medicine for treating ankylosing spondylitis The composition is for a medicinal liquor for treating ankylosing spondylitis. The raw materials used include: C. sativus, one adult Zoacys dhumnades, and a white liquor. This medicinal liquor can dispel cold and dampness, nourish kidney yang, improve blood circulation, and improve resistance through the compatibility of the C. sativus, the Z. dhumnades, and the white liquor. The efficacy rate of the medicinal liquor is 100% (Laiwang, 2014).

33.4.46 Externally-applied wet tissue for treating measles This invention aims to provide an externally-applied wet tissue for treating measles from the following medicinal materials: honeysuckle, C. sativus mint, radix Sophorae flavescentis, cortex dictamni (Dictamnus dasycarpus), brine alkali, roots of kudzu vines (Pueraria montana), Cimicifugae foetidae, and nonwoven fabrics. These materials have sterilizing and itch-relieving properties and can clear heat, promote diuresis, help eczema, and treat measles (Liu, 2015).

33.4.47 Treatment of herpes zoster The drug to treat herpes zoster is prepared from the combination of the following herbal medicines: pilea cavaleriei (Pilea cavaleriei), hainan ervatamia root, Kalanchoe laciniata, humifuse euphorbia herb (Euphorbia humifusa), herb of argun groundsel (Senecio argunensis), fishing pole, dayflower (Commelina communis), peruvian groundcherry herb (Physalis peruviana), philippine violet herb (Barleria cristata), Elephantopus scaber, shellac, herb of common achyranthes, C. sativus, Hylotelephium verticillatum, etc. This composition has positive effects such as clearing away heat and toxic materials, boosting blood circulation to remove blood stasis, relieving swelling and pain, discharging liver,

504

SECTION | VI Saffron and health

adjusting qi, and treating herpes zoster. Due to the use of “green” or environmentally friendly, and pollution-free natural drugs, this product is safe and effective with no toxicity or side effects. The drug is easy to use, simple to formulate and prepare, rapid in effect, and short in treatment duration (Xia and Zhenrong, 2015).

33.4.48 Chinese composition for treating septic shock This composition is the traditional Chinese medicine formula for treating septic shock and associated multiorgan dysfunction syndrome. It is comprised of red ginseng, radix aconiti lateralis preparata, Pulsatilla chinensis, L. wallichii, S. miltiorrhiza, radix aucklandiae (S. costus), radix ophiopogonis, and C. sativus. This formula has the beneficial effects of promoting blood circulation to remove blood stasis, clearing heat and toxicity, strengthening energy, and tonifying qi. This traditional Chinese medicine formula has accurate treatment effect, no toxicity or side effects, many clinical applications, and a total effective rate exceeding 85% (Yan et al., 2015).

33.4.49 Drug for treating bone injury This medicine for treating bone injury is comprised of C. sativus L., Notoginseng root, myrrha, frankincense, radix dipsaci, sappan wood, etc. This formula is prepared for curing numerous bone injuries such as pain from bruised bones, dislocation of bones, acute and chronic sprains, bone fractures, and comminuted fracture splits (Shaojiang, 2006).

33.4.50 Traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases This Chinese medicine for treating cardiovascular and cerebrovascular diseases is comprised of raw materials including: C. pilosula, pangolin, pearl, saffron crocus, tortoise plastron, cockroach, luffa seed (Luffa aegyptiaca), angelica, benevolence, etc. The medicine improves blood circulation to remove blood stasis, soothes liver, clears and activates the channels and collaterals, adjusts qi, tonifies spleen, nourishes yin and primordial spirit, and conditions triple burner. It can be used for perfecting the blood quality, improving blood circulation to remove meridian obstruction, softening blood vessels, strengthening the blood vessel elasticity, and removing necrotic cells. Moreover, the effective rate of the preparation is as high as 96% (Zhanming, 2015).

33.4.51 Medicine for treating eyelid eczema This medicine contains rubiaceae borreria stricta, C. sativus, tea begonia, Ficus simplicissima lour, multiflora rose fruit, Aristolochia moupinensis franch, Artemisia rupestris, virginia creeper (Parthenocissus quinquefolia), herba lycopi (L. lucidus var. hirtus), clematis fasciculifloora franch (Clematis fasciculiflora), paniculate swallowwort root (C. paniculatum), honeysuckle, radix rubiae (Rubia cordifolia), human placenta, and P. multiflorum. This medicine is prepared for the treatment of eyelid eczema. This product can eliminate stasis (by prompting blood circulation), clear toxins and heat, remove dampness, nourish blood, and wet dryness (Hefang, 2015a).

33.5

Food products

33.5.1 Vegetable drinks Healthy vegetable-based drinks contain raw materials such as saffron, jasmine, theanine, [gamma]-aminobutyric acid, and wolfberry, which lead to stress relief, improved quality of sleep, boosted immune system and gastrointestinal motility, and regulation of endocrine functions (Kun et al., 2017).

33.5.2 Healthy drink prepared from saffron pollen Saffron pollen-based drinks are extracted from saffron pollen essence through nano-scale ultrafine crushing. This healthy saffron drink provides many benefits by increasing the absorption of nutrients, supplying oxygen-rich blood in circulation, restoring skin health and beauty, regulating endocrine functions, and reducing stains caused by endocrine disorders (Chao, 2016). Available saffron formulations and patents are listed in Table 33.1.

TABLE 33.1 Available saffron formulations and patents. Patent number

Title

Composition

Route

References

CN103263373B

Saffron shower gel

Water citron fruit, safranin

External (gel)

Cheng et al. (2015)

Jilin white ginseng, placenta CN 1268318C

Face cream

Jilin white ginseng, placenta, saffron, pearl, white tuber, fresh white skin, white sandalwood, astragalus, angelica, bletilla musk, pluarity of Chinese herbs lanolin, and milk

External (cream)

Yaocheng (2006)

CN106137907A

Hand cream

Water, ethanol, propylene glycol, butanediol, isopropyl palmitate, cetostearyl alcohol, hyaluronic acid, carbomer, milk protein, tocopherol, allantoin, hydroxyethyl cellulose, DMDM hydantoin, PEG-7 glyceryl cocoate, PEG-40 hydrogenated castor oil, polysorbate-20, phenoxyethanol, chlorphenesin, glycerin, sodium polyacrylate, sodium benzoate, menthol PCA ester, bio-saccharide gum, evening primrose seed oil, avocado oil, olive-pomace oil, Blumea oil, spearmint leaf oil, ginseng extract, catnip leaf extract, pearl extract, herba centellae, immortelle extract, Oryza sativa oil, grape seed extract, aloe extract, tremella extract, propolis, donkey-hide gelatin, saffron crocus extract, rhodiola rosea extract, radix angelicae extract, tea extract, lemon juice, rose extract, alcohol extract of verbena officinalis, lavender extract, and salvia officinalis extract

External (cream)

Baoquan (2016)

CN101028239A

Biological cosmetic cream

Bee ova, grub, honey, saffron, and wine

External (cream)

Jitong et al. (2009)

CN101606899A

Freckle-whitening makeup

Ginseng, honeysuckle, saffron licorice, Sichuan Shao angelica glycerin, parts of Polygonum multiflorum white peony root, white parts friendly, white cover, white tuber, white parts eligible atractylodes, radix, white envy Li, fritillaria, ebony, subwire injustice, epimedium, herb parts, cyperus, and motherwort

External

Li (2009)

CN 105055272A

Vanishing cream

Aloe extracting solution, ginkgo biloba, honey, pearl powder, amomum tsao-ko, ginseng, saffron crocus, radix angelicae, fresh milk, essence, and deionized water

External

Zhenbiao (2015)

CN 104188880B

Repair cream

Collagen jelly, three peptides, red ginseng extract, uronic acid vitamin vitamin E coixenolide, squalene, grape seed extract, celery leaf extract, snow lotus extract, pea extract parts, clove leaf extract, peanut sprout extract, modified corn starch, purple sweet potato anthocyanin, cucumber extract, sophora extract, watermelon extract, pearl powder, lettuce extract, onion extract, cnidium extract, lotus root extract, and saffron extract

External

Yang (2016)

CN101744971B

Beautifying and removing

Saffron crocus, muskone, sage powder, pearl powder, radix paeoniae alba, aloe and, nopal

External (liquid cream)

Yuhong (2011)

CN 102370608A

Moisturizing and whitening

Wistaria sinensis flowers, magnolia flower, rose, almond, saffron, mythic fungus, ginseng, red ginseng, American ginseng, white peony root, rhizoma typhoon, and radix astragali

External (cream)

Yi (2012)

(Continued )

TABLE 33.1 (Continued) Patent number

Title

Composition

Route

References

CN103446019A

Whitening, elimination of freckles and improvement of elasticity of skin

Homemade rose dry, dry saffron, and water

External (mask)

Hao and Wei (2013)

CN104398426A

Antiacne

Saffron crocus, sophora japonica, bletilla licorice and radix angelicae, lavender essential oil, and rose essential oil

External

Chen et al. (2015)

CN103446037B

Whitening freckle

Safflower, rhodiola, mulberry, lactiflorae, atractylodes baiji, sweet almond, and white tuckahoe

External

Weimin (2016)

CN 104293542A

Acne-removing

Wormwood soaking solution, mugwort powder, evening primrose oil, tea, grape seed extract, pure water, vitamin E, olive oil, palm oil, coconut oil, laurel leaf, asarum, snow lotus, angelica, ginseng, cnidium, peach, saffron, ginkgo, biloba, garland chrysanthemum, cicada, qu beans, osmanthus, lemon, banana peel, wine, clove parts, puhuang, and loofah

External (soap)

Zhen (2015)

CN 101579300A

Lotion

Pasty attapulgite clay, saffron, fresh lotus leaves, fresh common cephalanoplos herb, honey, iso-amyl p-methoxycinnamate, rutile type titanium dioxide, butanediol, potassium sorbate, and deionized water

External

Wenling et al. (2009)

CN104523562A

Toothpaste

Abrasives, cleaning agents, wetting agents, adhesives, fragrances sweeteners, preservatives, tibet cordyceps, fritillaria, saffron, and water

External

Russian (2015)

CN 101357224B

Drug for kidney health

Aweto, ginseng like palm, snow lotus herb, saffron, Rhodiola rosea, kudzu root, epimedium, and superoxide dismutase

Oral (capsule)

Yucong and Kunming (2010)

US20160199398A1

Compositions containing enriched natural crocin and/or crocetin

Enriched or purified natural crocin or crocetin or combination of both and possible other active phytochemicals (quercetin, resveratrol, pterostilbene, curcumin, theaflavin, theaflavin-3,30 digallate, theaflavin-3-gallate, theaflavin-30 -gallate, anthocyanidins, anthocyanins, catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, nobiletin, tangeretin, α-lipoic acid, L-carnitine, carnosine, astragaloside, cycloastragenol, lutein, β-lycopene, zeaxanthin, rosarin, β-rosasterol, rosavin, rosin, dihydromyricetin, chlorogenic acid, glycyrrhizic acid ammonium salt, rebaudioside A, green tea extract, black tea extract, bilberry extract, ginseng extract, grape extract, grape seed extract, epimedium extract, schisandra extract, astragalus extract, rhodiola extract, citrus fruit extract, orange extract, lemon extract, licorice extract, artichoke leaf extract, gotu kola extract, and stevia extract)

Oral, parenteral, percutaneous, rectal, mucosal, intranasal, or topical administration

Gao (2016)

CN103040683A

Nourishing hair conditioner

Saffron crocus, P. multiflorum, licorice, honeysuckle, lecithin, casein, amino acid, radix stemonae, methyl parahydroxybenzoats cassia twig, essence and deionized water

External

Yan (2013)

CN1141805A

Angiitis, veno-occlusive disease of liver, osteomyelitis, and bone tuberculosis

Saffron, white pepper, cow’s periost, pearl powder, tortoise plastron glue, jujube, and Chinese angelica root

Not explained

Zuohua (2000) (Continued )

TABLE 33.1 (Continued) Patent number

Title

Composition

Route

References

83115

Depression, Alzheimer’s disease, sexual dysfunction, metabolic syndrome, diabetes, burning mouth syndrome, age-related macular degeneration, and cancer

Crocin, a carotenoid chemical compound and the main active constituent of saffron

Oral

Mohajeri et al. (2014)

CN102895426A

Treating rheumatic osteopathia

Saffron, peach kernel, angelica, peony root, asarum, angelica, clematis, Lycopodium clavatum, contact Ishido, haifengteng, chuanxiong, kusnezoffii, chuanwu, papaya, rhizoma, and sorghum liquor

External (topical spray)

Hai (2014a)

CN105287667A

Treating arthritis and rheumatism

Saffron, panax, ginger active ingredient, achyranthes, mint mixture comprising one or more extraction at an arbitrary ratio

External (liquid patch)

Ziqiang et al. (2016)

CN106620459A

Treating dermatitis

Rhizome of yunnan multileaf paris, root of integripetal rhodiola, rhizome of common burred, rhizome of glabrous greenbrier, rhizome of swordlike atractylodes, gan huang shui (sus.), root of tonkin sophora, snake slough, belvedere fruit, mulberry silkworm, rhizome of involucrate stahlianthus, Chinese brake herb, rhizome of common anemarrhena, saffron crocus stigma, raw fruit of Siberian cocklebur, and stem and leaf of tube fleece flower

External (ointment)

Jian (2017)

CN101987181A

Treating acne

Oldenlandia diffusa, honeysuckle, forsythia, rehmanniae, moutan bark, red paeonia, radix scutellariae, cortex mori radicis, gypsum, Salvia miltiorrhiza, spina gleditsiae, divaricate saposhnikovia root, licorice, saffron, folium eriobotryae, licorice, selfheal, coix seed, bryozoatum, brimstone, rhizoma coptidis, borneol, chloromycetin, radix sophorae falvescentis, frankincense, dandelion, and viola yedoensis

External (ointment)

Xiaorui (2011)

CN101045114A

Treating skin scar

Turtle shell, cibotium, angelica, codonopsis, saffron, borneol, ganoderma

External (spray)

Xiuyun (2007)

EP2070545A1

Prevention and treatment of inflammatory disorders of the colon

Mint essential oil, Curcuma longa derivatives, Olea europea derivatives, and possibly one or more of N-acetylcysteine, glutathione, ubidecarenone, lactoferrin, carotenoids, polyphenols, vitamin C, vitamin E, extract or derivative of St. John’s Wort, kava kava, saffron, valerian, passion-flower, chamomile, and griffonia

Oral

Tramonti (2009)

CN100548342C

Treating diabetes

Bear bile, emblica, saffron, artificial musk, barberry bark, licorice paste, ink Beijing, cassia, and betel nut

Different forms such as pills, powders, capsules, soft capsules, granules

Wei (2009)

CA2362606C

Treating skin inflammations and bacterial infections such as acne, pseudofolliculitis, local redness, and local odor with these compositions

Alcohol and/or triclosan in combination with phenoxyethanol, benzalkonium or benzethonium chloride, cocophosphatidyldimoniun chloride and plant extracts [preferably selected from C. longa, Crocus sativus (saffron), Alkanna tinctoria (henna root), and Hydrastis canadensis (golden seal)]

Antimicrobial compositions (e.g., gels)

Jampani et al. (2013)

(Continued )

TABLE 33.1 (Continued) Patent number

Title

Composition

Route

References

CN1081057C

Treating acute and chronic oral diseases

Saffron, zaopi, zao ren, wolfberry, arsenic and indigo gray, and borneol

Oral

Xiuzhen (2002)

CN103463597A

For treating gout

Saffron, safflower, leaves, loosestrife, natural coarse salt particles, pepper, ginger, and evening primrose

Oral

Wenju (2016)

CN102743517A

Prepregnancy fetus protection pills

Large head atractylodes rhizome, eucommia, Chinese eaglewood, saffron, dipsacus root, malaytea scurfpea fruit, baikal skullcap root, and selenium element

Oral

Jing (2012)

WO2011034528A1

Composition for relief of cold and flu symptomatic

Black pepper, cumin, and ginger. In at least one embodiment of the present invention, the formulation of the aqueous dietary supplement composition further includes turmeric, cinnamon, rose hip, and saffron

Not explained

Gopinathan (2011)

US8993008B1

Treating diabetes

Gymnema extract, bilberry extract, Asian ginseng extract, fenugreek extract, marshmallow extract, ceylon cinnamon extract, bitter melon extract, autumn crocus extract, bay laurel extract, colocynth extract, and prickly pear extract

Oral

Mashat Ba and Ihab (2015)

CN1098682C

Treating and prevention of cardiovascular disease

Saffron plurality pharmaceutically glycosides or saffron polyglycoside and a pharmaceutically acceptable excipient

Oral

Fengyun (2003)

CN 1883672A

Treating mouth ulcers

Saffron, safflower, Ligusticum wallichii, bitter bamboo leaf, belamcanda rhizome, and calcarea lime

External

Fengyun (2006)

US20100028464A1

Satiety agent for treatment of obesity

Safranal and/or picrocrocin and/or crocin and/or derivatives

Oral

Bourges (2010)

CN104042942 A

Treatment of chronic obstructive pulmonary disease in stable phase

Codonopsis, Chinese yam, asters, tinglizi, acanthopanax, epimedium before, maodongqing, white, gold boiling grass, nepeta, angelica, river willow, dogwood, scrophulariaceae, kelp, bamboo garden, millettia, Chinese caterpillar fungus, black sesame, suddenly real, rehmannia, one hundred vine, bitter almonds, France breit, gray meat rong, jujube, orange peel, toosendan, shidi, saffron, hematoxylin, north grass, and ligusticum

Oral

Feng and Wenkui (2014)

CN1333028 A

Rheumatic arthritis

Krait/agkistrodon, black-striped snake, centipede, salvia root, ephedra, large gentian root, notoginseng, saffron, etc.

External (topical)

Shuren (2002)

CN105497335A

Drug for treating rheumatic arthritis

Cassia twig, longhairy antenoron herb, semen strychni, divaricate saposhnikovia root, processed manis pentadactyla, common andrographis herb, cortex acanthopanacis, saffron, notopterygium root, burdock, fructus forsythiae, pubescent angelica root, dandelion, garden balsam stem, suberect spatholobus stem, mint, papaya, mulberry twig, radix et rhizoma rubiae, harlequin glorybower leaf, gentian, and radix pleurospermi tibetanic

Not explained

Huile (2016)

CN100423743C

Medicine for treating duodenal bulbar ulcer or inflammation

Licorice, safflower, red dates, honey, licorice, and saffron

Oral

Kong et al. (2007) (Continued )

TABLE 33.1 (Continued) Patent number

Title

Composition

Route

References

CN103550520B

Traditional Chinese medicinal preparation for treating gastric

Saffron, bai caoshuang, mango nuclear, malan, beans, bamboo pepper, aeschynomene, hungry leeches, kowloon root, good vine, turnip, onion skin, and revive rattan grass

Oral

Guorong and Zejin (2014)

CN1055002C

Cream for treatment of breast cancer

Saffron, astragalus root, forsythia fruit, burreed tuber, zedoary, curcuma root, angelica, cactus, and huang bai

External

Zhenfeng (2000)

CN1213561A

Drug for treating brain apoplexy

Saffron, baizhu, leech peony shichangpu, salvia chuanxiong, achyranthes large bezoar, chicken blood Teng Teng, peach angelica, safflower guizhi, shanjia beads astragalus wind, silkworm scorpion, hematoxylin motherwort, and jatamansi sunburn strychnos powder

Not explained

Jixu (2000)

83169

Reduce Alzheimer’s disease symptoms

Crocin

Oral

Mafi (2014)

US20170106038A1

Prevention and/or treatment of degenerative eye disorders

Saffron and curcumin

Oral

Bisti et al. (2017)

CN102743525A

Traditional Chinese medicinal preparation for treating prostatic hyperplasia caused by kidney qi deficiency

Radix astragali, arillus longan, rhizoma Atractylodis macrocephala, fructus corni, cortex moutan, S. miltiorrhiza, Acorus gramineus, radix bupleuri, Pyrrosia lingua, combined spicebush root, mulberry fruit, wolfberry fruit, Glechoma longituba, spica prunellae, white peony root, cassia twig from szechwan, C. sativus, Hedyotis diffusa, Medulla Junci, cinnamon, carapax testudinis, and rhizoma alismatis

Oral

Shuhua (2012)

CN103751470A

Healthcare oil for treating gynecological diseases

Olive oil, C. sativus flower, P. multiflorum, Poria cocos, Angelica sinensis, Cyperus rotundus, Spatholobus suberectus stem, Caesalpinia sappan, and herba Asari

External (topical)

Hai (2014b)

CN102526415B

Chinese medicine for treating dysmenorrhea

Salvia, vinegar fumarate, saffron, white wine shao, wine angelica, march, motherwort, cyperus, toosendan, bupleurum, and honey-roasted cumin licorice

Oral

Mei (2013)

CN104940885A

Chinese medicine for treating dysfunction of endocrine

Fineleaf schizonepeta herbs, moxa liquoric roots, white muscardine silkworm, notopterygium roots, balloon flowers, Chinese thorowax roots, Cremastra appendiculata, piperis longi, Lycopus lucidus, flying bandit, oriental wormwood, Geranium strictipes, dendrobium, roselle, Leonurus macranthus, mioga ginger, C. sativus, herba hyperici japonici, Panax notoginseng, heartleaf houttuynia herbs, Coriolus versicolor, Chinese angelica, bighead atractylodes rhizome, white peony roots, cinnamon, Morinda officinalis, Chinese magnoliavine fruit, male silkworm moths, sliced aconite, male cloves, fossil fragments, polygonatum, jujubes, alismatis, P. cocos, and root bark of the peony tree

Oral

Yueqin (2015)

CN104587188A

Drug for treating premature ovarian failure

Boschniakia rossica, Lethariella zahlbruckneria, gorse, sparrow, licorice and lanatechead saussurea herb with flower, polyalthia root, Chinese date, Panax japonicas, Itea chinensis hook, Pleurotus citrinopileatus sing, Muraenesox cinereus, Polygonatum kingianum, Ventilago leiocarpa benth, cucubalus root, and C. Sativus

Not explained

Ping et al. (2015)

(Continued )

TABLE 33.1 (Continued) Patent number

Title

Composition

Route

References

CN104352890A

Traditional Chinese medicine for treating rheumatic heart disease

Radix glycyrrhizae preparata, dried tangerine peel, cassia twigs, Codonopsis pilosula, C. sativus, radix paeoniae rubra, Astragalus membranaceus, Radix aconiti lateralis preparata, rhizoma atractylodis, cortex acanthopanacis and Coix lachrymal. C. sativus in this product has an important role in treatment of stagnation, xiongge nausea, typhoid mad, trance afterward, vomiting, amenorrhea, irregular menstruation blood stagnation, postpartum lochia, bleeding ached, measles, and bruises

Oral

Jianli (2015)

CN104383465A

Chinese medicine for treating cataract

Butterfly bush flower, selfheal, black soybean flower, semen cassia, fructus lycii, Scrophularia ningpoensis, radix curcumae, ternate grape fern herb, hollow azurite, Marsh equisetum, C. sativus, boa gall, littletooth hydrangeavine root, and Cardamine lyrata

Oral

Hefang (2015b)

CN103550716A

Medicine for treatment of cervical spondylosis

A. sinensis, the rhizome of chuanxiong, radices sileris peach kernel, C. pilosula, A. membranaceus, pawpaw, caulis spatholobi, ground beetle, radix clematidis, root of bidentate achyranthes, parasitic loranthus, notopterygium root, radix curcumae, C. sativus, and asarum

Not explained

Fen (2014)

CN104042822A

Chinese medicine preparation for treating qi stagnation and blood stasis amenorrhea

Mulberry, nothopanax, Salvia evansiana, Salvia bowleyana, Salvia chinensis, C. sativus, sinkiang corydalis tuber, Callosciurus erythraeus powder, all grass of sinuate tickclover, folium apocyni veneti, Jasminum grandiflorum, rhizoma cyperi, fingered citron, and licorice

Oral

Li (2014)

CN101947291B

Composition for treating osteoproliferation and herniated disk

Wine-fried Chinese angelica, divaricate saposhnikovia root, C. sativus, vinegar-processed pangolin scales, dragon’s blood, root of red-rooted salvia, wine-fried rhubarb, vinegar-processed corydalis tuber, vinegar-processed frankincense, myrrh, medicinal cyathula root, scalded drynaria rhizome, asarum, sand-scalded eucommia bark, incised notopterygium rhizome, heracleum, tall gastrodia tuber, obscured homalomena rhizome, schizophragma integrifolium, lumbricus, fried insect, clematis root, processed rhizoma atractylodis, and liquoric root

Oral

Shichang et al. (2012)

CN104645030A

For treating blocking-antibody-deficiency type recurrent spontaneous abortion

Human placenta, herba epimedii, M. officinalis, radix astragali, ginseng, semen cuscutae, stir-fried radix paeoniae alba, stirfried rhizoma A. macrocephala, licorice, male silkworm moth, rhizoma polygonati, corn cervi pantotrichum, Cynomorium songaricum, herba cistanche, gecko, walnut, rhizoma curculiginis, radix P. multiflorum preparata, fructus lycii, stirfried rhizoma dioscoreae, P. cocos, radix pseudostellariae, C. sativus, chinese angelica, and radix salviae miltiorrhizae

Oral

Enxue (2012)

(Continued )

TABLE 33.1 (Continued) Patent number

Title

Composition

Route

References

CN103784753A

For treating nasopharyngitis

Scaphium scaphigerum, Platycodon grandiflorum, radix scrophulariae, herba rabdosiae, dried radix rehmanniae, Rabdosia rubescens, Dendrobium nobile, American ginseng, mint, radix ophiopogonis, isatis root, olive, momordica grosvenori, tendril-leaved fritillary bulb, cordyceps sobolifera, C. sativus, and qingxiangcao

Oral

Ji et al. (2014)

CN104013792A

For treating lung tumor

Saffron, cordyceps, figwort, ophiopogon, licorice, honeysuckle, rose root, herb parts, and ganoderma

Oral

Chen and Wang (2014)

CN104208332A

For treating damp-heat stagnation type abdominal mass

Berberine, acid-slip, megan colored parts, society finch, tieshu, cicada insects, palmetto sub, saffron, bell capillaris, verbena, ganoderma, and licorice

Oral

Sun (2014)

CN104758829A

For treating nonulcer dyspepsia

Epimedium, curculigo, poria, medlar, angelica, shao parts, atractylodes, licorice, codonopsis, habitat, saffron, cordyceps, cyperus, turmeric, huang jing, dodder, dendrobium, and pseudo-ginseng

Oral

Guohua (2015)

CN104667138A

For treating nonulcer dyspepsia

Epimedium, curculigo, poria, medlar, angelica, shao parts, atractylodes, licorice, codonopsis, habitat, saffron, cordyceps, cyperus, turmeric, huang jing, dodder, dendrobium, and pseudo-ginseng

Oral

Shulin (2015)

CN104547542A

For treating peptic ulcer

Radix bupleuri, Pistacia lentiscus, medicated leaven, C. sativus, lignum sappan, Polygonatum sibiricum, Malus baccata, fritillary, radix glycyrrhizae, rhizoma corydalis, P. notoginseng, C. rotundus, Cynanchum paniculatum, Oreorchis patens, radix aucklandiae, myrrh, rhizoma coptidis, fennel seeds, cacumen platycladi, folium artemisiae argyi, and sarcodactylis

Oral

Zhang et al. (2015)

CN103690696A

For treating ankylosing spondylitis

C. sativus, one adult Zoacys dhumnades, and white liquor

Oral

Laiwang (2014)

CN104435413A

Externally-applied wet tissue for treating measles

Honeysuckle, mint, radix Sophorae flavescentis, cortex dictamni, brine alkali, shellac, C. sativus, roots of kudzu vines selaginella tamariscina, Cimicifugae foetidae, and nonwoven fabrics

External (topical wipes)

Xingwang (2015)

CN104784496A

For treating herpes zoster

Vegetable oil, a single timber, kalanchoe, humifusa cut long grass, fishing rods, commelina, planterns, boil poisonous weeds, bitter gall, dried comfrey, grass upside down, saffron, round leaf eight, stone see through, snow-capped mountains sage, blue flame, andrographis, hair musk, will be saved, sedum thirty-seven snake backward, baiyao son, ma buildings fruit, hemp quartet, august sapporo, juju, lindera, fu paper cutters, small flying grass, belvedere fruit, wheat bran grass, and licorice

External (ointment)

Wang and Zhao (2015)

CN104435669A

For treating septic shock and accompanying multiorgan dysfunction syndrome

Red ginseng, aconite, takes office, salvia, pulsatilla, woody parts, radix, and saffron

Oral (decoction, granules, capsules, tablets, pellets)

Yan et al. (2015) (Continued )

TABLE 33.1 (Continued) Patent number

Title

Composition

Route

References

CN1814106A

For treating bone injuries

Saffron, thirty-seven frankincense and myrrh, akagi chuan-off double flower, and kowloon’s blood

Oral (solution)

Shaojiang (2006)

CN104721680A

For effectively treating cardiovascular and cerebrovascular diseases

Codonopsis, pangolin, pearl, saffron, turtle shell, earthworm, treating diabetes, cockroaches, the subgourd, angelica, wan renxin, nosed pit viper, and S. miltiorrhiza

Oral (capsules)

Zhanming (2015)

CN104491311A

Medicine for treating eyelid eczema

Rubiaceae borreria stricta, C. sativus, tea begonia, Ficus simplicissima lour, multiflora rose fruit, Aristolochia moupinensis franch, Artemisia rupestris, virginia creeper, herba lycopi, clematis fasciculifloora franch, paniculate swallowwort root, honeysuckle, radix rubiae, human placenta, and P. multiflorum

Oral

Hefang (2015a)

CN106923115A

Relieve stress, get better quality of sleep, promote the immune systems, gastrointestinal motility, and regulation of endocrine system

Saffron, jasmine, theanine, [gamma]-aminobutyric acid, and wolfberry

Oral

Kun et al. (2017)

CN105433374A

Supply of oxygen blood circulation, restore beauty skin healthy, regulation of endocrine system, reduction of stains caused by endocrine disorders

Saffron pollen

Oral

Chao (2016)

Available saffron formulations and product patents Chapter | 33

33.6

513

Conclusion

Several findings highlight the function of saffron and its derivatives in a number of patents and formulations. Saffron constituents are patented in various drug preparations of poly herbal formulations for the treatment of cardiovascular and central nervous system diseases. Saffron-based products are also used in the promotion of immune function and treatment of depression. Importantly, the observed synergistic effects of saffron with other substances that are found in these herb combinations preparations indicate that the curative effect is not attributed only to saffron.

References Aliakbarzadeh, G., Parastar, H., Sereshti, H., 2016. Classification of gas chromatographic fingerprints of saffron using partial least squares discriminant analysis together with different variable selection methods. Chemom. Intell. Lab. Syst. 158, 165
173. Baoquan, L., 2016. Hand Cream. China Patent Application CN106137907A. Bisti, S., Maccarone, R., Anna Maggi, M., 2017. Compositions Based on Saffron for the Prevention and/or Treatment of Degenerative Eye Disorders. United States Patent Application US20170106038A1. Bourges, C., 2010. Use of Saffron and/or Safranal and/or Crocin and/or Picrocrocin and/or Derivatives Thereof as a Sateity Agent for Treatment of Obesity. United States Patent Application US20100028464A1. Chao, L., 2016. Healthy Drink Prepared From Saffron Pollen. China Patent Application CN105433374A. Chen, Y., Wang, C., 2014. Traditional Chinese Medicine for Treating Lung Tumor and Preparation Method Thereof. China Patent Application CN104013792A. Chen, Y., Dong, Q., Huaying, F., 2015. Mask for Treating Common Acne. China Patent Application CN104398426A. Cheng, J., Jingjia, S., Yuan, W., Xiaodong, H., 2015. One Kind of Saffron Shower Gel and Preparation Method. China Patent Application CN103263373A. Dong, Z., Xiuxian, H., Xue, L., 2015. Traditional Chinese Medicinal Preparation for Treating Peptic Ulcer and Method for Preparing Preparation. China Patent Application CN104547542A. Enxue, T., 2012. Traditional Chinese Medicine for Treating Blocking-Antibody-Deficiency Type Recurrent Spontaneous Abortion. China Patent Application CN101947291A. Fen, W., 2014 Traditional Chinese Medicine for Treating Cervical Spondylosis. China Patent Application CN103550716A. Feng, W., Wenkui, W., 2014 Medicine for Treating Chronic Obstructive Pulmonary Disease at Stable Phase. China Patent Application CN104042942A. Fengyun, H., 2003. Pharmaceutical Process of Crocus sativus Polyglycoside and Its Tablets. China Patent Application CN1237419A. Fengyun, H., 2006. Externally Applied Chinese Medicine for Treating Ulcerative Stomatitis. China Patent Application CN1883672A. Gao, S., 2016. Compositions Containing Enriched Natural Crocin and/or Crocetin, and their Therapeutic or Nutraceutical Uses. United States Patent Application US20160199398A1. Gopinathan, G., 2011. Cold and Flu Symptomatic Relief Composition. France Patent Application WO2011034528A1. Guohua, L., 2015. Traditional Chinese Medicine Composition for Clinically Nursing and Treating Ascites due to Cirrhosis. China Patent Application CN104758829A. Guorong, Y., Zejin, Z., 2014. Traditional Chinese Medicinal Preparation for Treating Gastric Ulcer and Preparation Method Thereof. China Patent Application CN103550520A. Hai, H., 2014a. Externally Applied Spraying Agent for Treating Rheumatic Osteopathia. China Patent Application CN102895426A. Hai, L., 2014b. Healthcare Oil for Treating Gynecologic Diseases and Preparation Method Thereof. China Patent Application CN103751470A. Hao, L., Wei, L., 2013. Self-Made Rose Mask. China Patent Application CN103446019A. Hefang, C., 2015a. Medicine for Treating Eyelid Eczema And Application Thereof. China Patent Application CN104491311A. Hefang, C., 2015b. Traditional Chinese Medicine Preparation for Treating Cataract and Preparation. China Patent Application CN104383465A. Hosseini, S.I., Farrokhi, N., Shokri, K., Khani, M.R., Shokri, B., 2018. Cold low pressure O2 plasma treatment of Crocus sativus: an efficient way to eliminate toxicogenic fungi with minor effect on molecular and cellular properties of saffron. Food chem. 257, 310
315. Huile, X., 2016. Chinese Medicinal Formula for Preventing and Treating Rheumatic Arthritis. China Patent Application CN105497335A. Jampani, H., Ellis, T., Newman, J., 2013. Therapeutic Antimicrobial Compositions. Canada Patent Application CA2362606A1. Ji, X., S, Y., Zhang, L., 2014. Chinese Herbal Medicine Formula for Treating Nasopharyngitis and Preparation Method Thereof. Chinese Patent Application CN103784753A. Jian, C., 2017. Ointment for Treating Dermatitis. China Patent Application CN106620459A. Jianli, H., 2015. Traditional Chinese Medicine Preparation for Treating Rheumatic Heart Disease. China Patent Application CN104352890A. Jing, L., 2012. Pre-Pregnancy Fetus Protection Pills. China Patent Application CN102743517A. Jitong, H., Yalei, H., Jingtao, H., Kuiian, L., Junya, L., 2009. Biological Cosmetic Cream With Spot-Dispelling Function and Its Production. China Patent Application CN101028239A. Jixu, Z., 2000. Medicine for Treating Brain Apoplexy. China Patent Application CN1213561A. Kong, L., Wang, X., Zhao, Z., Ingzhen, Y., Lanjuan, X., Yingdong, K., 2007. Medicine for Treating Duodenal Bulbar Ulcer or Inflammation. China Patent Application CN1899409A.

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Kun, L., Xiaohui, S., Yahong, Z., Yongtong, A., Guan, Y., Yanjun, L., 2017. Plant Beverage Containing Saffron as Well as Preparation Method and Application of Plant Beverage. China Patent Application CN106923115A. Laiwang, Z., 2014 Medicinal Liquor for Treating Ankylosing Spondylitis, and Processing Method Thereof. China Patent Application CN103690696A. Li, L., 2009 Traditional Chinese Medicine Freckle Whitening Makeup. China Patent Application CN101606899A. Li, S., 2014. Traditional Chinese Medicine Capable of Treating Qi Stagnation and Blood Stasis Amenorrhea. China Patent Application CN104042822A. Liu, X., 2015. Externally-Applied Wet Tissue for Treating Measles and Preparation Method Thereof. China Patent Application CN104435413A. Mafi, F., 2014. Preparation of Saffrotin From Saffron for Prevention and Treatment of Alzheimer’s disease. Iran Patent Application 83169. Mashat Ba, H., Ihab, A., 2015. Herbal Composition for Treating Diabetes. United States Patent Application US8993008B1. Mei, L., 2013. Chinese Medicinal Composition for Treating Dysmenorrhea and Preparation Method of Same. China Patent Application CN102526415A. Mohajeri, S., Tabassi, S., Hadizadeh, F., Badinloo, M., 2014. Preparation of Crocin Tablet (Krocinas). Iran Patent Application 83115. Ping, S., Jufeng, W., Jian, L., Bo, D., 2015. Formula of Traditional Chinese Medicine for Treating Premature Ovarian Failure. China Patent Application CN104587188A. Russian, F., 2015. Tibetan Worm Grass, Fritillaria and Saffron Crocus Toothpaste. China Patent Application CN104523562A. Shaojiang, Z., 2006. Medicine for Treating Bone Injures and Preparing Method. China Patent Application CN1814106A. Shichang, H., Jinshuai, H., Jintao, H., 2012. Chinese Medicinal Composition for Treating Osteoproliferation and Herniated Disk and Preparation Method Thereof. China Patent Application CN101947291A. Shuhua, S., 2012. Traditional Chinese Medicinal Decoction for Treating Prostatic Hyperplasia Caused by Kidney Qi Deficiency. China Patent Application CN102743525A. Shulin, W., 2015. A Traditional Chinese Medicine for Treating Nonulcer Dyspepsia. China Patent Application CN104667138A. Shuren, Z., 2002 External Liniment for Treating Rheumatic Arthritis. China Patent Application CN1333028A. Sun, L., 2014. Traditional Chinese Medicine for Treating Damp-Heat Stagnation Type Abdominal Mass. China Patent Application CN104208332A. Tramonti, P., 2009. Oral Compositions for the Prevention and Treatment of Inflammatory Disorders of the Colon. European Patent Office EP2070545A1. Wang, X., Zhao, Z., 2015. Drug for Nursing and Treating Herpes Zoster and Preparation Method of Drug. China Patent Application CN104784496A. Wei, L., 2009. Night Oral Administration Tibet Medicine for Treating Diabetes and Its Preparing Method. China Patent Application CN1899472A. Weimin, S., 2016. MOISTURE Preparation and Medicine Mask Freckle. China Patent Application CN103446037A. Wenju, Z., 2016. Health-Care Medicine for Treating Gout. China Patent Application CN103463597A. Wenling, Z., Wenlan, J., Qinghua, X., Shengying, X., 2009. Attapulgite Saffron Suntan Lotion. China Patent Application CN101579300A. Xia, W., Zhenrong, Z., 2015 Drug for Nursing and Treating Herpes Zoster and Preparation Method of Drug. China Patent Application CN104784496A. Xiaoqing, J., Yamen, S., Ling, Z., Xiaoxiao, X., 2014. China Herbal Medicine Formula for Treating Nasopharyngitis and Preparation Method Thereof. China Patent Application CN103784753A. Xiaorui, W., 2011. Chinese Medicine Ointment for Treating Acne. China Patent Application CN101987181A. Xingwang, L., 2015. Externally-Applied Wet Tissue for Treating Measles and Preparation Method Thereof. China Patent Application CN104435413A. Xiuyun, K., 2007. External Use Spray for Treating Skin Scar. China Patent Application CN101045114A. Xiuzhen, S., 2002. Chinese Medicine Composition for Treating Oral Disease and Preparing Method Thereof. China Patent Application CN1234263A. Yan, C., Xiao, L., Hengdong, Z., Xi, Z., 2015. Traditional Chinese Medicine Composition for Treating Septic Shock and Accompanying Multiorgan Dysfunction Syndrome. China Patent Application CN104435669A. Yan, W., 2013. Traditional Chinese Medicine Nourishing Hair Conditioner and Preparation Method Thereof. China Patent Application CN103040683A. Yang, L., 2016. Fu Yang Repair Cream. China Patent Application CN104188880A. Yaocheng, Y. 2006 Face Cream for Retaining Youthful Looks. China Patent Application CN1561955A. Yi, Z., 2012. Fair Complexion Powder for Face and Preparation Method Thereof. China Patent Application CN102370608A. Yucong, Z., Kunming, Z., 2010. Kidney-Tonifying and Health-Preserving Preparation. China Patent Application CN101357224B. Yueqin, Y., 2015 Traditional Chinese Medicine for Treating Dysfunction of the Endocrine. China Patent Application CN104940885A. Yuhong, Y., 2011 Beautifying and Beverage Removing Combined Liquid and Preparation Method Thereof. China Patent Application CN101744971A. Yulian, C., Chaobin, W., 2014 Traditional Chinese Medicine for Treating Lung Tumor and Preparation Method Thereof. China Patent Application CN104013792A. Zhang, D., Hao, X., Xue, L., 2015. Traditional Chinese Medicinal Preparation for Treating Peptic Ulcer and Method for Preparing Preparation. China Patent Application CN104547542A. Zhanming, J., 2015. Traditional Chinese Medicine for Effectively Treating Cardiovascular and Cerebrovascular Diseases and Preparation Method Thereof. China Patent Application CN104721680A. Zhen, Y., 2015. Herbal Essential Oil Acne-Removing Soap. China Patent Application CN104293542A. Zhenbiao, L., 2015. Aloe Vanishing Cream Capable of Removing Spots and Whitening. China Patent Application CN105055272A.

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Zhenfeng, N., 2000 External Use Cream for Treatment of Breast Cancer. China Patent Application CN1158262A. Ziqiang, Z., Yujian, L., Xuemin, W., 2016. Traditional Chinese Medicine Liquid Patch for Treating Arthritis and Rheumatism. China Patent Application CN105287667A. Zuohua, P., 2000. Chinese Medicine for Treatment of Angiitis. China Patent Application CN1141805A.

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Chapter 34

Safety and toxicity of saffron Soghra Mehri1, Bibi-Marjan Razavi1 and Hossein Hosseinzadeh2 1

Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, 2Pharmaceutical

Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

Chapter Outline 34.1 Introduction 34.2 Experimental data on the safety and toxicity of saffron and its bioactive ingredients in animal models 34.2.1 Acute toxicity 34.2.2 Subacute toxicity 34.2.3 Subchronic and chronic toxicity

34.1

517 518 518 518 519

34.2.4 Developmental toxicity 34.2.5 Mutagenicity and genotoxicity 34.3 Clinical studies on safe and toxic doses of saffron and its bioactive ingredients 34.4 Conclusion References

520 521 523 527 528

Introduction

Saffron is the most expensive dietary spice and is derived from the stigmas of Crocus sativus L. Most of this herbal medicine is produced in Iran, mainly in South Khorasan province (Alavizadeh and Hosseinzadeh, 2014; Shahi et al., 2016). The taste and odor of this spice is due to picrocrocin and safranal, while the red color is due to the crocin (Alavizadeh and Hosseinzadeh, 2014; Shahi et al., 2016). The carotenoids of saffron are sensitive to light, heat, oxygen, and enzymatic oxidization (Hosseini et al., 2018a). Saffron has a long history of use in traditional medicine (Hosseinzadeh and Nassiri-Asl, 2013; Mollazadeh et al., 2015). In various studies, saffron and its main constituents have exhibited different valuable properties (Alavizadeh and Hosseinzadeh, 2014; Hosseini et al., 2018b; Hosseinzadeh and Jahanian, 2010; Hosseinzadeh et al., 2008; Khazdair et al., 2015; Shafiee et al., 2017). Experimental studies have confirmed the positive effects of saffron and its active ingredients in cardiovascular disorders (Broadhead et al., 2016; Imenshahidi et al., 2010), convulsion (Hosseinzadeh and Talebzadeh, 2005; Khazdair et al., 2015), depression (Broadhead et al., 2016; Khazdair et al., 2015; Vahdati-Hassani et al., 2014), Alzheimer’s (Alavizadeh and Hosseinzadeh, 2014; Broadhead et al., 2016; Khalili and Hamzeh, 2010), cancer (Aung et al., 2007; Abdullaev and Espinosa-Aguirre, 2004), metabolic syndrome (Doumouchtsis et al., 2018), renal failure (Boozari and Hosseinzadeh, 2017), and digestive disorders (Khorasany and Hosseinzadeh, 2016). Additionally, the therapeutic effects of saffron in depression (Shafiee et al., 2017), Alzheimer’s (Akhondzadeh et al., 2010a), and diabetic maculopathy (Sepahi et al., 2018) have been reported in clinical studies. Interestingly, saffron and its bioactive ingredients have been mentioned as protective or antidote components against natural or chemical toxins (Razavi and Hosseinzadeh, 2015). The protective effects of these agents against potent toxins including bisphenol A (Vahdati-Hassani et al., 2017), diazinon (Lari et al., 2013; Razavi et al., 2013a,b), malathion (Mohammadzadeh et al., 2018), acrylamide (Mehri et al., 2015), cumene hydroperoxide (Yousefsani et al., 2018), and doxorubicin (Chahine et al., 2016; Elsherbiny et al., 2016) have been confirmed. Although saffron-based compounds have been extensively investigated in pharmacological, clinical, and toxicological aspects, little is known about the safety of these agents in vivo (Badie-Bostan et al., 2017), so classified information about the toxicity and safety of this valuable herbal medicine is needed.

Saffron: Science, Technology and Health. DOI: https://doi.org/10.1016/B978-0-12-818638-1.00035-6 © 2020 Elsevier Inc. All rights reserved.

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In the current chapter, we categorize the toxicity and safety of saffron, crocin, crocetin, picrocrocin, and safranal into two parts: in the first part, basic data about the toxicity of these agents in animal models are discussed. In the second part, we focus on the safety and adverse effects of saffron, which havev been studied in clinical trials.

34.2 Experimental data on the safety and toxicity of saffron and its bioactive ingredients in animal models 34.2.1 Acute toxicity In acute toxicity tests, determination of the LD50 value (the amount of the substance required to kill 50% of the test population) is the main goal. A LD50 value can be affected by route of administration, strain, age, sex, type of feed, and weight of the animals (Eaton and Gilbert, 2015). Based on the LD50 values in acute toxicity tests, the chemical agents can be classified with different toxicity levels: supertoxic (,1 mg kg21), extremely toxic (,5 mg kg21), highly toxic (1
50 mg kg21), moderately toxic (50
500 mg kg21), slightly toxic (500
5000 mg kg21), practically nontoxic (5000
15,000 mg kg21), and relatively harmless ( . 15,000 mg kg21). In this section we considered the LD50 of saffron, crocin, and safranal to evaluate toxicity. Different studies have reported saffron LD50 values: 1. Following oral administration of aqueous extract of saffron in BALB/c mice, the LD50 value was 4120 6 556 mg kg21 (Bahmani et al., 2014). 2. Intraperitoneal (IP) exposure of saffron stigma extract in mice resulted in an LD50 value of 1.6 g kg21 (Karimi et al., 2004). 3. Following IP administration of saffron petal extract in mice, LD50 value 6 g kg21 was found (Karimi et al., 2004). 4. Oral administration of ethanolic saffron extract in mice up to 5 g kg21 did not produce any detectable acute toxic effects or deaths (Ramadan et al., 2012). According to classification of chemicals based on LD50 values, it can be concluded that saffron extract is slightly toxic and practically nontoxic in acute exposure. Administration of crocin (0.5, 1, 1.5, 2, and 3 g kg21, IP or orally 3 g kg21) did not show any mortality after 24 and 48 hours of treatment in mice. Regarding the tolerated dose of 3 g kg21 of crocin (orally and IP administration), it has been found to be practically low toxic in acute ingestion or IP treatment (Hosseinzadeh et al., 2010). The acute toxicity of safranal was evaluated in rat and mice within 2 days after IP or oral administration. The LD50 values of safranal were 1.48 mL kg21 in male mice, 1.88 mL kg21 in female mice, and 1.50 mL kg21 in male rats following IP exposure, while oral LD50 values were 21.42 mL kg21 in male mice, 11.42 mL kg21 in female mice, and 5.53 mL kg21 in male rats. According to this study, safranal was low toxic in acute IP exposure and practically nontoxic in acute oral administration in both mice and rats. Differences between oral and IP LD50 values in part can be due to the effects of first-pass metabolism and low absorption following oral treatment (Hosseinzadeh et al., 2013).

34.2.2 Subacute toxicity In subacute toxicity tests, the toxicity of a chemical agent after repeated administration is studied. Biochemical parameters and histopathological findings are evaluated after either 14 or 28 days of exposure. The obtained data is used for dose selection in prolonged studies (Eaton and Gilbert, 2015). Daily IP administration of aqueous extract of stigma (0.16, 0.32, and 0.48 g kg21) and petal (1.2, 2.4, and 3.6 g kg21) for 2 weeks markedly reduced RBC (red blood cell), HCT (hematocrit), Hb (hemoglobin), and induced anemia in rat. The aqueous extract of petal in 2.4 and 3.6 g kg21 doses elevated the level of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and LDH (lactate dehydrogenase) enzymes. Also, liver and lung injuries were observed following treatment by stigma extract (Karimi et al., 2004). Repeated IP administration of ethanolic extract of saffron (0.35, 0.7, and 1.05 g kg21) for 2 weeks to rats reduced RBC, HCT, and Hb and increased WBC (white blood cell) counts and the levels of ALT and AST enzymes. Additionally, the level of serum uric acid, urea, and creatinine were significantly elevated. Based on the pathological studies, the ethanolic extract of saffron was found to induce mild-to-severe renal and hepatic injuries (Mohajeri et al., 2007). In another study, oral administration of saffron at a dose of 200 mg kg21 for 28 days could markedly decrease spermatogenesis index in rats (Khayatnouri et al., 2011).

Safety and toxicity of saffron Chapter | 34

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Intraperitoneal administration of aqueous extract of saffron (200 mg kg21) three times per week for 4 weeks to rats increased the level of ALT, uric acid, and WBC (Hariri et al., 2018). Saffron petal ethanolic extract (0, 75, 150, 225, and 450 mg kg21) was injected intraperitoneally to rats for 14 days. No significant changes in hematological parameters and spleen histology were observed. The level of IgG at dose of 75 mg kg21 was significantly elevated in comparison to other groups (Babaei et al., 2014). Overall, it can be concluded that anemia, liver, lung, and renal injuries are the main findings in subacute toxicity of saffron, especially in high doses. In order to evaluate the hepatotoxicity of crocin in rat, different doses of this carotenoid (50, 100, and 200 mg kg21) were administrated IP once a week for 4 weeks. At the end of treatment, antioxidant enzymes including catalase, superoxide dismutase, and glutathione peroxidase (GPx) and serum biomarkers such as AST, ALP, ALT, uric acid, urea, and creatinine were measured. In addition, induction of oxidative stress was evaluated through measurement of protein carbonyl and malondialdehyde levels and glutathione content. No significant changes in serum parameters and oxidative stress biomarkers were observed. Also, histopathological analysis showed that crocin at these doses could not induce liver injury. The activity of GPx was reduced at higher dose of crocin (200 mg kg21) (Taheri et al., 2014). In another study, rats were treated with crocin (15, 45, 90, and 180 mg kg21, IP) for 21 days. Biochemical, hematological, and histopathological parameters as well as changes in weight and amount of food intake were measured in this study. According to the results, weight loss, reduction in food intake, decline in platelets and creatinine levels, elevation of LDL levels, reduction in alveolar size in lungs, myosin light chain atrophy, and decrease in size of cell nuclei in heart were the main findings following exposure to crocin (180 mg kg21) (Hosseinzadeh et al., 2010). Administration of crocin (50 mg kg21, IP) for 4 weeks to rats did not induce cardiotoxicity, which was evaluated through oxidative stress assay, histopathological examination, and apoptosis markers analysis (Razavi et al., 2013a). Subacute administration of crocin (20 mg kg21) did not induce hepatotoxicity in rats. The normal level of liver biomarkers including ALT and AST, normal histopathological pattern of liver, no significant alteration in the level of mitogen-activated protein kinase (MAPK) pathway proteins, normal content of GSH and of 8-iso prostaglandine F2a confirmed crocin at this dose and duration of exposure could not induce liver injury (Vahdati-Hassani et al., 2017). Regarding the obtained results, it can be concluded that subacute treatment with crocin at these doses does not induce significant organ toxicity. The subacute oral administration of safranal (0.1, 0.25, or 0.5 mL kg21 daily for 21 days) markedly reduced RBC, Hb, HCT, and platelets and did not show any significant alteration in some biochemical markers including total bilirubin, serum glucose, albumin, serum creatinine, ALT, AST, CPK (creatine phosphokinase), and total bilirubin in rats. The levels of total cholesterol, triglyceride, and ALP were significantly reduced with 0.5 and 0.25 mL kg21 doses of safranal. Also, significant elevation in the levels of LDH and serum urea nitrogen (BUN) was observed at higher dose of safranal. Safranal (0.5 mL kg21) induced noticeable abnormalities in lung and kidneys. The main findings were edema and cytolysis in kidney, progressed emphysema, and lymphocyte infiltration in lungs (Hosseinzadeh et al., 2013). Toxic effects of safranal (0.1, 0.5, and 1 mL kg21, IP for 21 days) on humoral and cellular immune system of mice have been investigated. The results showed that safranal at studied doses and duration of exposure does not have immunotoxic properties (Riahi-Zanjani et al., 2015). Overall it seems that subacute exposure to safranal at high doses induces toxicity.

34.2.3 Subchronic and chronic toxicity A subchronic toxicity test will determine the toxic effects following repeated administration of a substance during 30
90 days. In this type of study, mortality, body weight, food/water consumption, hematology, urinalysis, organ weights, and histopathology are measured. Generally, NOAEL (NO observed adverse effect level) and LOAEL (lowest observed adverse effect level) are determined during this test. The obtained data of subchronic tests are used basically for dose selection in chronic studies (Eaton and Gilbert, 2015). Chronic toxicity tests are designed to evaluate the toxic effects as a result of more than 3 months of exposure. The duration of exposure is somewhat dependent on the intended period of exposure in humans. The purpose of this type of study is the assessment of physiological, pathological, pharmacokinetic, and carcinogenicity aspects of the substance (Eaton and Gilbert, 2015). In order to determine the subchronic effects of saffron, the ethanolic extract (500 mg kg21, orally) was administrated to rats for 8 weeks. According to the results, saffron did not affect the serum activity of ALT and AST or the serum

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levels of creatinine and urine. Other parameters including food consumption, food conversion ratio, and body weight gain were also not changed. But relative weights of kidneys and liver significantly increased in treated rats (Ramadan et al., 2012). Following oral administration of aqueous saffron extract (120 mg kg21) for 105 days, no significant changes in metabolic parameters, organ weight, and levels of ALT and AST were observed (Lahmass et al., 2017). There is less information about subchronic and chronic toxic effects of saffron and its main constituents, so the design of studies aimed at evaluating such effects are needed. Although according to the mentioned reports, no significant toxicity was observed at tested doses in subchronic exposure to saffron.

34.2.4 Developmental toxicity Developmental toxicology explains any structural malformation, functional abnormality, and growth retardation caused by toxic chemicals or physical factors. It is the study of adverse effects on the development of the organism following exposure to toxic agents in different periods including before conception, during prenatal development, or postnatally until puberty (Carney et al., 2011; Eaton and Gilbert, 2015). Due to the widespread use of herbal products, it is critical to understand how herbal medicines may affect embryonic development. Different studies have been conducted to investigate the effects of saffron and its main constituents on fetal development. In order to investigate the teratogenic effects of saffron, pregnant BALB/c mice received different doses of aqueous saffron extract (0.2%, 0.4%, and 0.8%) in whole gestational period and third trimester throughout the gestational period. The results showed that placental weight and diameter, body and tail length, mean fetal weight, and biparietal diameter significantly decreased in animals treated with a higher dose of solution and mostly following exposure during the whole gestational period. Also, the mean number of resorbed and dead fetuses increased in a dose-dependent manner (Zeynali et al., 2009). Administration of saffron in the first or second trimester of the gestational period in mice elevated the mean number of resorbed and dead fetuses. The maximum resorbed fetuses were recorded in animals receiving 0.8% saffron solution during the first trimester while the maximum dead fetuses were observed in animals receiving higher doses of saffron during the second trimester (Hosseini et al., 2009). In another study, saffron (2.5 and 100 mg kg21) was administrated to pregnant mice for 5 days during the first and second weeks of gestation and for 4 days during the third week of gestation. Embryonic toxicity and congenital malformations were evaluated on days 14 and 18 of gestation and day 1 neonates. Both doses of saffron led to significant reduction in embryonic growth parameters. Congenital malformations were observed in treated embryos and neonates including subcutaneous bleeding and head malformations (Al-Qudsi and Ayedh, 2012). Oral administration of saffron (50, 250, and 1000 mg kg21) on day 5 until day 19 of gestation in Wistar rat did not affect maternal body weight gains, uterine weight, food intake, corpora lutea, and implantation counts, pre- and postimplantation loss, litter size, weight, and length. Also, soft tissue abnormalities and skeletal malformation were not observed (Edamula et al., 2014). In order to determine postnatal developmental toxicity of saffron, pregnant Wistar rats were treated with saffron (50, 250, and 1000 mg kg21, orally) from implantation (day 5 postcoitus) through lactation up to lactation day (LD) 20. Results showed that maternal/lactation body weight gains, food intake, and fertility were unaffected. Also, saffron at tested doses did not exhibit any adverse effects on the mean number of pups born and weight of male and female pups and pup survivability. It can be concluded that saffron did not cause maternal toxicity or toxicity on the developing fetus/pups (Edamula et al., 2014). The embryonic toxicity of the main constituents of saffron was investigated following IP administration of crocin (200 and 600 mg kg21) or safranal (0.075 and 0.225 mL kg21) on gestational days 6
15. The length and weight of fetuses markedly reduced in animals receiving crocin or safranal. Additionally, minor skeletal abnormalities, growth retardation, mandible, and calvaria malformations were the main findings in these groups (Moallem et al., 2013). In another study, the effects of crocetin (10, 25, 50, 100, and 200 μM) on Xenopus embryos were evaluated and compared with teratogenic effects of all-trans retinoic acid (ATRA). Neither crocetin nor ATRA exhibited embryolethal effects at the tested doses. Head-to-tail length and eye diameters reduced in all doses of crocetin while cement gland length decreased in higher doses. Crocetin appears to be 236 times less potent at decreasing the length of frog embryo, 371 times less potent at reducing eye diameter, and 397 times less potent at decreasing cement gland size in comparison to ATRA. Clearly, crocetin showed much fewer teratogenic effects than ATRA in all tested parameters (Martin et al., 2002). As can be seen, saffron and its main constituents exhibited teratogenic effects particularly at high doses.

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34.2.5 Mutagenicity and genotoxicity Genotoxicity testing is generally performed to determine the destructive effects of substances on the genetic material of cells (DNA, RNA). Different in vivo and in vitro tests including genotoxicity bacterial reverse mutation test (Ames test), mammalian bone marrow chromosome aberration test, micronucleus test, mouse heritable translocation assay, and in vitro sister chromatid exchange assay in mammalian cells have been used to evaluate genotoxicity (Jena et al., 2002; Saks et al., 2017). The antimutagenic and comutagenic effects of saffron and its main constituents were evaluated using the Ames/ Salmonella test system and two well-known mutagens BP (benzo[a]-pyrene) and 2AA (2-amino-antracene). Saffron extract (100
1500 μg per plate) did not induce genotoxic effects using the Salmonella typhimurium tester strain TA98, both with and without S9 activation, but comutagenic effect of saffron on 2-AA-induced mutagenicity dosedependently was detected. Crocin and picrocrocin at different concentrations (100
400 nM) per plate exhibited no comutagenic effects, while safranal at these doses markedly induced the comugagenic property on 2-AA-induced mutagenicity. It appears that the comutagenic effect of saffron is related to safranal (Abdullaev et al., 2003). Crocetin did not show genotoxic effect in V79 cells, which was determined using the Ames test, rec-assay, and sister chromatid exchange test (Ozaki et al., 2002). Single IP administration of safranal (5 mL kg21) to NMRI mice did not induce DNA migration in liver, kidney, and spleen organs, as determined by comet assay (Hosseinzadeh and Sadeghnia, 2007). Administrations of saffron extract (50, 100, and 200 mg kg21 IP, three times per week) (Hariri et al., 2018) or crocin (50, 100, and 200 mg kg21, IP, three times per week) did not elevate micronucleus indices (Hariri et al., 2011), while treatment of rats with safranal (0.1 mL kg21, IP, three times per week) significantly increased micronucleus index as a marker of genotoxicity (Hariri et al., 2011). It can be concluded that safranal at high doses can induce genotoxicity. Experimental studies related to the safety and toxicity of saffron, crocin, and safranal are summarized in Tables 34.1
34.3, respectively. TABLE 34.1 Experimental studies related to safety and toxicity of saffron. Toxicity

Route, dose, and duration of exposure

Animal model

Results

References

Acute

Oral administration of aqueous extract of saffron

BALB/c mice

LD50: 4120 6 556 mg kg21

Bahmani et al. (2014)

IP exposure of saffron stigma extract

Male Wistar rat

LD50: 1.6 g kg21

Karimi et al. (2004)

IP exposure of saffron petal extract

Male Wistar rat

LD50: 6 g kg21

Karimi et al. (2004)

Oral administration of ethanolic saffron extract

Male mice

No detectable acute toxic effects or deaths up to 5 g kg21

Ramadan et al. (2012)

IP, aqueous extract of stigma (0.16, 0.32, 0.48 g kg21), 2 weeks

Male Wistar rat

Reduction of RBC, HCT, Hb, and induction anemia in rat

Karimi et al. (2004)

Subacute

Liver and lung injuries IP, aqueous extract of petal (1.2, 2.4, 3.6 g kg21) g kg21, 2 weeks

Male Wistar rat

Reduction of RBC, HCT, Hb, and induction anemia in rat

Karimi et al. (2004)

Elevation of ALT, AST, and LDH levels in higher doses IP, ethanolic extract (0.35, 0.7, 1.05 g kg21) 2 weeks to rats

Male Wistar rat

Reduction of RBC, HCT, Hb Elevation of WBC, ALT, AST, urea, uric acid, and creatinine

Mohajeri et al. (2007)

Mild-to-severe hepatic and renal injuries Oral, saffron 200 mg kg21 for 28 days

Male rat

Reduction of spermatogenesis index

Khayatnouri et al. (2011) (Continued )

TABLE 34.1 (Continued) Toxicity

Route, dose, and duration of exposure

Animal model

Results

References

IP, aqueous extract of saffron (200 mg kg21), three times per week for 4 weeks

Male Wistar rat

Elevation of WBC, ALT, and uric acid

Hariri et al. (2018)

IP, Saffron petal ethanolic extract (0, 75, 150, 225, and 450 mg kg21) for 14 days

Male Wistar rat

No significant changes in hematological parameters and spleen histology were observed

Babaei et al. (2014)

Elevation the level of IgG at dose of 75 mg kg21 Subchronic

Oral, ethanolic extract of saffron (500 mg kg21), for 8 weeks

Male sprague dawley rat

No significant changes in AST, ALT, creatinine, urine, food consumption, food conversion ratio, and body weight gain

Ramadan et al. (2012)

Relative weights of kidneys and liver significantly increased

Developmental toxicity

Oral, aqueous extract of saffron (120 mg kg21), for 105 days

Male Wistar rat

No significant changes in metabolic parameters, organ weight, ALT, and AST levels

Lahmass et al. (2017)

Oral, aqueous saffron extract (0.2%, 0.4%, 0.8%) 1st or

Pregnant BALB/c mice

Reduction of placental weight and diameter, mean fetal weight, body and tail length, and biparietal diameter

Hosseini et al. (2009)

2nd trimesters throughout the gestational period Oral, aqueous saffron extract (0.2%, 0.4%, 0.8%) in whole gestational period and 3rd trimester throughout the gestational period

Elevation of the mean number of resorbed and dead fetuses Pregnant BALB/c mice

Reduction of placental weight and diameter, mean fetal weight, body and tail length, and biparietal diameter

Zeynali et al. (2009)

Elevation of the mean number of resorbed and dead fetuses

Genotoxicity

Oral, aqueous saffron extract (2.5, 100 mg kg21) to for 5 days during the 1st and 2nd weeks of gestation and for 4 days during the 3rd week of gestation

Pregnant mice

Reduction of embryonic growth parameters subcutaneous bleeding, and head malformations

Al-Qudsi and Ayedh (2012)

Oral, saffron (50, 250, 1000 mg kg21), during day 5 until day 19 of gestation

Pregnant Wistar rat

No changes in maternal body weight gains, food intake, uterine weight, corpora lutea, implantation counts, pre- and postimplantation loss, litter size, weight, and length. No soft tissue abnormalities and skeletal malformation.

Edamula et al. (2014)

Oral, saffron (50, 250, 1000 mg kg21) from implantation (day 5 postcoitus) through lactation up to LD 20

Pregnant Wistar rats

No changes in maternal/lactation body weight gains, food intake, no adverse effects on the mean number of pups born and weight of male and female pups and pup survivability

Edamula et al. (2014)

Oral, aqueous saffron extract (50, 100, 200 mg kg21) three times per week for 4 weeks

Male Wistar rat

No changes in micronucleus index

Hariri et al. (2018)

Saffron extract (100
1500 μg plate21)

Ames/ Salmonella typhimurium test

No genotoxic effect

Abdullaev et al. (2003)

Comutagenic effect on 2-aminoantracene-induced mutagenicity

ALT, Alanine aminotransferase; AST, aspartate aminotransferase; Hb, hemoglobin; HCT, hematocrit; IP, intraperitoneal; LDH, lactate dehydrogenase; RBC, red blood cell; WBC, white blood cell.

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TABLE 34.2 Experimental studies related to safety and toxicity of crocin. Toxicity

Dose, route, and duration of exposure

Animal model

Results

References

Acute

IP (0.5, 1, 1.5, 2, 2.5, 3 g kg21)

Male razi mice

Tolerated dose: 3 g kg21

Hosseinzadeh et al. (2010)

Female Wistar albino

No significant changes in serum parameters and oxidative stress biomarkers

Taheri et al. (2014)

Orally (3 g kg21) Subacute

IP (50, 100 and 200 mg kg21) once a week for 4 weeks

No liver injury Reduction of GPx activity at 200 mg kg21

IP (15, 45, 90, 180 mg kg21) for 21 days

Male Wistar rat

Weight loss, reduction in food intake, decline in, platelets and creatinin levels, elevation of LDL level, reduction in alveolar size in lungs, myosin light chain atrophy, decrease in size of cells nuclei in heart were the main findings following exposure to crocin (180 mg kg21)

Hosseinzadeh et al. (2010)

IP (50 mg kg21) for 4 weeks

Male Wistar rat

No changes in oxidative stress, histophatological, and apoptotic markers in heart

Razavi et al. (2013a)

IP (20 mg kg21) for 4 weeks

Male Wistar rat

No changes in oxidative stress, biochemical and MAPK proteins in liver

VahdatiHassani et al. (2017)

Developmental toxicity

IP (200, 600 mg kg21) on gestational days 6
15.

Pregnant mice

Reduction of length and weight of fetuses

Moallem et al. (2013)

Genotoxicity

100
400 nM plate21

Ames/ Salmonella typhimurium test

No comutagenic effect on on 2-amino-antraceneinduced mutagenicity

Minor skeletal abnormalities, growth retardation, mandible, and calvaria malformations

Abdullaev et al. (2003)

GPx, Glutathione peroxidase; IP, intraperitoneal; LDL, low-density lipoprotein; MAPK, mitogen-activated protein kinase.

34.3

Clinical studies on safe and toxic doses of saffron and its bioactive ingredients

Saffron has been utilized as a food additive without complications for several centuries. According to some studies, doses up to 1.5 g day21 are believed to be safe and adverse effects are reported with doses more than 5 g day21 with lethal dose of approximately 20 g. It has been reported that doses more than 10 g day21 have been used for induction of abortion. Some adverse effects including vomiting, uterus bleeding, hematuria, bleeding of the gastrointestinal mucosa as well as vertigo and dizziness have been reported at this dose (Schmidt et al., 2007). The safety of saffron has been directly reported in few studies. For example, in healthy volunteers, crocin (20 mg day21) for 1 month did not show any clinically important adverse effects in comparison with placebo, although reductions in serum amylase, mixed WBCs, and PTT were observed (Mohamadpour et al., 2013). For evaluating the safety of saffron, a double-blind, placebo-controlled study with healthy adult volunteers was performed. Saffron tablets (400 mg day21, for 1 week) did not show any significant adverse effects on blood pressure except for reducing standing systolic blood pressure and mean arterial pressures (Modaghegh et al., 2008). In addition, some hematological parameters such as RBCs, hemoglobin, hematocrit, and platelets were reduced slightly by saffron tablets (200 and 400 mg, for 1 week). Saffron also elevated sodium, blood urea nitrogen, and creatinine. These alterations were in normal ranges and were not clinically important (Modaghegh et al., 2008).

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TABLE 34.3 Experimental studies related to safety and toxicity of safranal. Toxicity

Dose, route, and duration of exposure

Animal model

Results

References

Acute

IP

Male BALB/c mice

LD50: 1.48 mL kg21

Hosseinzadeh et al. (2013)

IP

Female BALB/c mice

LD50: 1.88 mL kg21

Hosseinzadeh et al. (2013)

IP

Male Wistar rats

LD50: 1.50 mL kg21

Hosseinzadeh et al. (2013)

Oral

Male BALB/c mice

LD50: 21.42 mL kg21

Hosseinzadeh et al. (2013)

Oral

Female BALB/c mice

LD50: 11.42 mL kg21

Hosseinzadeh et al. (2013)

Oral

Male Wistar rats

LD50: 5.53 mL kg21

Hosseinzadeh et al. (2013)

Oral (0.1, 0.25, or 0.5 mL kg21) daily for 21

Male Wistar rats

Reduction of RBC, Hb, HCT, and platelets

Hosseinzadeh et al. (2013)

Subacute

Reduction of total cholesterol, triglyceride, and ALP in 0.5 and 0.25 mL kg21 doses Elevation of the level of LDH and BUN, kidney and lung abnormalities at 0.5 mL kg21 IP (0.1, 0.5, 1 mL kg21) for 21 days

Male BALB/ cimice

No immunotoxic effects

Riahi-Zanjani et al. (2015)

Developmental toxicity

IP, (0.075 and 0.225 mL kg21) on gestational days 6
15

Pregnant mice

Reduction of length and weight of fetuses

Moallem et al. (2013)

Genotoxicity

100
400 nM plate21

Ames/ Salmonella typhimurium test

Minor skeletal abnormalities, growth retardation, mandible, and calvaria malformations Comutagenic effect on on 2-aminoantracene-induced mutagenicity

Abdullaev et al. (2003)

ALP, Alkaline phosphatase; BUN, blood urea nitrogen; Hb, hemoglobin; HCT, hematocrit; LDH, lactate dehydrogenase; RBC, red blood cell; WBC, white blood cell.

In another clinical trial, the safety of saffron and crocin in patients with schizophrenia was evaluated. Toxic effects on thyroid, liver, kidney, and hematologic systems were not observed by saffron and crocin (15 mg twice daily) (Mousavi et al., 2015). The results of this study provides evidence for the safety of saffron. Consumption of saffron capsules (250 mg) in the active phase of the first stage of labor reduced the intensity of pain from 97.4 6 2.9 in placebo group to 85.9 6 8.4 in treated females, and did not show toxicity in infants and mothers (Ahmadi et al., 2014). Moreover, administration of three 250 mg saffron pills in 24 hours to women with a gestational age of 39
41 weeks increased the readiness of the cervix in term pregnancies without any reported adverse effects (Sadi et al., 2016). In another double-blind, placebo-controlled study, 1 week treatment with saffron tablets (200 and 400 mg day21) did not show any adverse effects on the coagulant and anticoagulant system (Ayatollahi et al., 2014). According to a meta-analysis of randomized controlled trials to evaluate the efficacy and safety of saffron in the treatment of major depressive disorder (MDD) compared to placebo and synthetic antidepressants, saffron was

Safety and toxicity of saffron Chapter | 34

525

considered as a safe drug without serious adverse events (Yang et al., 2018). In these studies, the stigma and petal of saffron [capsules of 30 mg day21 (BID)] for 4, 6, and 8 weeks were effective in the treatment of mild-to-moderate depression without any important adverse effects (Akhondzadeh-Basti et al., 2007; Akhondzadeh et al., 2005, 2004; Ghajar et al., 2017; Moshiri et al., 2006; Noorbala et al., 2005; Shahmansouri et al., 2014). In addition, the efficacy and safety of saffron in the treatment of patients with mild-to-moderate Alzheimer’s disease were evaluated. Patients randomly received saffron capsules (15 mg twice per day) for 22 (Akhondzadeh et al., 2010b) or 16 (Akhondzadeh et al., 2010a) weeks. The effect of saffron was compared to donepezil (Akhondzadeh et al., 2010b) or placebo (Akhondzadeh et al., 2010a). According to the results, saffron at this dose acted similar to donepezil in the treatment of mild-to-moderate Alzheimer’s disease after 22 weeks and considerably improved cognitive function more than placebo after 16 weeks. The frequency of adverse effects was not significant between saffron and placebo or donepezil groups. Only vomiting occurred significantly more frequently in the donepezil group (Akhondzadeh et al., 2010a,b). In a randomized, double-blind, placebo controlled clinical trial study, the safety of coadministration of saffron and SSRI on sexual dysfunction in patients with MDD was evaluated. Saffron (15 mg twice daily) for 4 weeks did not change significantly laboratory parameters such as liver and kidney function tests, blood cell counts, and coagulation tests (Mansoori et al., 2011). Moreover, in another clinical study conducted on patients with anxiety and depression, a 50 mg saffron capsule (Crocus sativus L. stigma) twice daily for 12 weeks showed a significant impact on the treatment of anxiety and depression disorder without any side effects (Mazidi et al., 2016). The safety of saffron extract (Affron, 14 mg BID) which was given to teenagers aged 12
16 years with mild-to-moderate anxiety or depressive symptoms for 8 weeks was evaluated. Saffron improved anxiety and depressive symptoms. Between placebo and saffron extract groups no significant adverse effects were observed. Only increased frequency of headaches in the placebo group compared to saffron group was observed (Lopresti et al., 2018). Furthermore, Affron (28 mg day21) for 4 weeks increased mood and decreased anxiety in healthy adults without side effects (Kell et al., 2017). To evaluate the safety of long-term or excessive crocetin consumption in healthy adult volunteers, physical examination, blood biochemistry, hematology, and urine were performed. The results showed that taking a crocetin capsule (7.5 mg per capsule) once per day for 12 consecutive weeks or 37.5 mg day21 for 4 consecutive weeks was safe (Yamashita et al., 2018a,b). To evaluate the effect of crocin on diabetic maculopathy, a doubleblind, placebo controlled, phase 2 randomized clinical trial was done. The results showed that administration of crocin 15 mg day21 for 3 months could significantly decrease HbA1c and central macular thickness and improve best-corrected visual acuity compared to the placebo group. Some side effects including increased appetite, subconjunctival hemorrhage, swelling, redness, and burning of the eyes associated with pain in some cases were reported. No statistical significant difference was observed between groups for the side effects discussed earlier (Sepahi et al., 2018). Additionally, the safety and efficacy of saffron (30 mg day21, BID) for 10 weeks in the treatment of mild-tomoderate obsessive-compulsive disorder were proven in another clinical study. Saffron was as effective as fluvoxamine (100 mg day21), and the frequency of adverse effects was not significantly different between the two groups (Esalatmanesh et al., 2017). Daily consumption of saffron (30 mg) reduced the severity of mild-to-moderate postpartum depressive disorder with no significant adverse effects on breastfeeding mothers or infants. Some adverse effects such gastrointestinal disorders, lack of sleep or oversleeping, low breast milk, and bleeding gums were observed in breastfeeding mothers that were not significantly important (Tabeshpour et al., 2017). Another double-blind, randomized clinical trial showed that consumption of saffron capsules (15 mg capsule) twice daily for 6 weeks is a safe alternative medication for reducing symptoms of postpartum depression. For patients taking fluoxetine (20 mg capsule) more headache, dry mouth, daytime drowsiness, constipation, and sweating side effects than the saffron group were reported that were not significantly important (Kashani et al., 2017). To evaluate the effects of saffron on the immune system, one 100 mg saffron tablet daily for 6 weeks was administrated to healthy men. Saffron elevated the IgG level and reduced the IgM level, the percentage of basophils and the platelets count, but increased the monocytes percentage after 3 weeks. After 6 weeks, these parameters returned to baseline levels. No adverse effects were reported (Kianbakht and Ghazavi, 2011). In summary, the common effective doses of saffron used in clinical practice (30
50 mg day21) were noticeably lower than toxic doses ( . 5 g day21), so saffron has a wide therapeutic index (Table 34.4).

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TABLE 34.4 Clinical studies related to safety of saffron. Study design

Dose/ constituents

Duration of exposure

Adverse effects

References

Double-blind, placebocontrolled study in the healthy adult volunteers

Crocin (20 mg day21)

1 month

k Serum amylase, mixed WBCs, PTT

Mohamadpour et al. (2013)

Double-blind, placebocontrolled study in the healthy adult volunteers

Saffron tablets (200 and 400 mg day21)

1 week

k Standing SBP, MAP

Modaghegh et al. (2008)

k RBCs, Hb, HCT, platelets m Na, BUN, Cr

Double-blind, placebocontrolled study was performed on patients with schizophrenia

Saffron aqueous extract and crocin (15 mg day21, BID)

12 weeks

No toxic effects on thyroid, liver, kidney, and hematologic systems

Mousavi et al. (2015)

Double-blind, placebocontrolled trial

Saffron capsule (250 mg)

Active phase of the first stage of labor

No significant adverse effect

Ahmadi et al. (2014)

Double-blind, placebocontrolled trial

Saffron (250 mg TID21)

24 hours

No significant adverse effect

Sadi et al. (2016)

Double-blind, placebocontrolled study

Saffron tablet (200 and 400 mg day21)

1 week

No adverse effect on coagulant and anticoagulant system

Ayatollahi et al. (2014)

Double-blind, randomized and placebo-controlled trial

Saffron capsule (30 mg day21, BID)

6 weeks

No significant differences in saffron and placebo groups in terms of the side effects

Akhondzadeh et al. (2005)

Double-blind, placebocontrolled and randomized trial

Saffron petal capsule (30 mg day21, BID)

6 weeks

No significant differences in saffron and placebo groups in terms of the side effects

Moshiri et al. (2006)

Randomized double-blind parallel-group study on depressed patients

Saffron capsule (30 mg day21, BID)

6 weeks

No significant differences between saffron and fluoxetine groups in the frequency of adverse events

Shahmansouri et al. (2014)

Double-blind randomized trial on depressed outpatients

Saffron capsule (30 mg day21, BID)

8 weeks

No significant differences between saffron and fluoxetine groups in the frequency of adverse events

AkhondzadehBasti et al. (2007)

Double-blind, randomized trial on depressed patients

Hydroalcoholic extract of saffron capsule (30 mg day21, BID)

6 weeks

No significant differences between saffron and fluoxetine groups in the frequency of adverse events

Noorbala et al. (2005)

Double-blind, randomized trial on depressed patients

Saffron capsule (30 mg day21, TDS)

6 weeks

Dry mouth and sedation were observed in imipramine group significantly more frequently than the saffron group

Akhondzadeh et al. (2004)

Double-blind, randomized trial on depressed patients

Saffron capsule (30 mg day21, BID)

6 weeks

No significant differences between saffron and citalopram groups in the frequency of adverse events

Ghajar et al. (2017)

Double-blind, randomized trial on patients with AD

Saffron capsule (30 mg day21, BID)

22 weeks

Vomiting occurred significantly more frequently in the donepezil group than in the saffron group

Akhondzadeh et al. (2010b)

Double-blind, randomized trial on patients with AD

Saffron capsule (30 mg day21, BID)

16 weeks

No significant differences between saffron and placebo groups in the frequency of adverse events

Akhondzadeh et al. (2010a) (Continued )

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527

TABLE 34.4 (Continued) Study design

Dose/ constituents

Duration of exposure

Adverse effects

References

Double-blind, placebo controlled clinical trial on depressed patients

Saffron capsule (30 mg day21, BID) and SSRI

4 weeks

No significant differences between saffron and placebo groups in the frequency of adverse events

Mansoori et al. (2011)

Double-blind, randomized, placebo controlled trial on patients with anxiety and depression

Saffron capsule (50 mg, BID)

12 weeks

Side effects were rare

Mazidi et al. (2016)

Randomized, double-blind, placebo-controlled trial on teenagers with anxiety or depressive symptoms

Saffron extract (Affron, 14 mg BID)

8 weeks

Headache occurred more frequently in the placebo group than in the saffron group

Lopresti et al. (2018)

Randomized, double-blind and placebo-controlled trial on healthy adults

Saffron extract (Affron, 14 mg BID)

4 weeks

No adverse effect

Kell et al. (2017)

Randomized, double-blind, placebo-controlled trial

Crocetin capsule (7.5 mg day21)

12 weeks

No adverse effect

Yamashita et al. (2018b)

Randomized, double-blind, placebo-controlled trial

Crocetin capsule (37.5 mg day21)

4 weeks

No adverse effect

Yamashita et al. (2018a)

Double-blind, placebo controlled, phase 2 randomized clinical trial on diabetic patients

Crocin (15 mg day21)

3 months

No significant differences between crocin and placebo groups in the frequency of adverse events

Sepahi et al. (2018)

Double-blind, randomized trial on patients with obsessivecompulsive disorder

saffron (30 mg day21, BID)

10 weeks

No significant differences between saffron and fluvoxamine groups in the frequency of adverse events

Esalatmanesh et al. (2017)

Double-blind, randomized and placebo-controlled trial on women with postpartum depressive disorder

Saffron (30 mg day21)

No significant differences between saffron and placebo groups in the frequency of adverse events

Tabeshpour et al. (2017)

Double-blind, randomized clinical trial on women with postpartum depressive disorder

Saffron (15 mg, BID)

6 weeks

No significant differences between saffron and fluoxetine groups in the frequency of adverse events

Kashani et al. (2017)

Randomized double-blind placebo-controlled clinical trial on healthy men

Saffron tablet (100 mg day21)

3 weeks

m IgG level

Kianbakht and Ghazavi (2011)

k IgM level k % basophils k Platelet counts m % monocytes

BUN, Blood urea nitrogen; Cr, creatinine; Hb, hemoglobin; HCT, hematocrit; MAP, mean arterial pressure; PTT, partial thromboplastin time; RBC, red blood cell; SBP, systolic blood pressure; WBC, white blood cell.

34.4

Conclusion

Different animal and clinical studies have been carried out to evaluate the toxicity and safety of saffron. In experimental studies, according to LD50 values, saffron, crocin, and safranal were found to be low or nontoxic agents. Exposure to high doses of saffron and its ingredients can induce organ and embryonic toxicity. Saffron at high doses may increase miscarriage rate in pregnant females, so using high doses of saffron during pregnancy should be avoided. According to some clinical reports saffron has a wide therapeutic index. Finally, as discussed in the chapter, the therapeutic doses of saffron in both clinical and experimental studies did not induce significant toxicity.

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References Abdullaev, F., Espinosa-Aguirre, J., 2004. Biomedical properties of saffron and its potential use in cancer therapy and chemoprevention trials. Cancer Detect. Prev. 28, 426
432. Abdullaev, F., Riveron-Negrete, L., Caballero-Ortega, H., Herna´ndez, J.M., Perez-Lopez, I., Pereda-Miranda, R., et al., 2003. Use of in vitro assays to assess the potential antigenotoxic and cytotoxic effects of saffron (Crocus sativus L.). Toxicol. In Vitro 17, 731
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10. Akhondzadeh, S., Fallah-Pour, H., Afkham, K., Jamshidi, A.H., Khalighi-Cigaroudi, F., 2004. Comparison of Crocus sativus L. and imipramine in the treatment of mild-to-moderate depression: a pilot double-blind randomized trial [ISRCTN45683816]. BMC. Compl. Alter. Med. 4, 12. Available from: https://doi.org/10.1186/1472-6882-4-12. Akhondzadeh, S., Tahmacebi-Pour, N., Noorbala, A.A., Amini, H., Fallah-Pour, H., Jamshidi, A.H., et al., 2005. Crocus sativus L. in the treatment of mild-to-moderate depression: a double-blind, randomized and placebo-controlled trial. Phytother. Res. 19, 148
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390.

Safety and toxicity of saffron Chapter | 34

529

Hosseini, A., Razavi, B.M., Hosseinzadeh, H., 2018b. Saffron (Crocus sativus) petal as a new pharmacological target: a review. Iran J. Basic Med. Sci. 21, 1091
1099. Hosseini, S., Dashti, M., Anvari, M., Zeynali, F., Miresmaeli, S., 2009. Studing teratogenic and abortification effects of different doses of saffron (Crocus sativus) decoction in 1st or 2 nd trimester in mice. Int. J. Reprode. Biomed. 7. Hosseinzadeh, H., Jahanian, Z., 2010. Effect of Crocus sativus L. (saffron) stigma and its constituents, crocin and safranal, on morphine withdrawal syndrome in mice. Phytother. Res. 24, 726
730. Hosseinzadeh, H., Nassiri-Asl, M., 2013. Avicenna’s (Ibn Sina) the canon of medicine and saffron (Crocus sativus): a review. Phytother. Res. 27, 475
483. Hosseinzadeh, H., Sadeghnia, H.R., 2007. Effect of safranal, a constituent of Crocus sativus (saffron), on methyl methanesulfonate (MMS)-induced DNA damage in mouse organs: an alkaline single-cell gel electrophoresis (comet) assay. DNA Cell Biol. 26, 841
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951. Hosseinzadeh, H., Sadeghi-Shakib, S., Khadem Sameni, A., Taghiabadi, E., 2013. Acute and subacute toxicity of safranal, a constituent of saffron, in mice and rats. Iran J. Pharm. Res. 12, 93
99. Imenshahidi, M., Hosseinzadeh, H., Javadpour, Y., 2010. Hypotensive effect of aqueous saffron extract (Crocus sativus L.) and its constituents, safranal and crocin, in normotensive and hypertensive rats. Phytother. Res. 24, 990
994. Jena, G., Kaul, C., Poduri, R., 2002. Genotoxicity testing, a regulatory requirement for drug discovery and development: Impact of ICH guidelines. Indian J. Pharmacol. 34 (2), 86
99. Karimi, G., Taiebi, N., Hosseinzadeh, H., Shirzad, F., 2004. Evaluation of subacute toxicity of aqueous extract of Crocus sativus L. stigma and petal in rats. J. Med. Plants 4, 29
35. Kashani, L., Eslatmanesh, S., Saedi, N., Niroomand, N., Ebrahimi, M., Hosseinian, M., et al., 2017. Comparison of saffron versus fluoxetine in treatment of mild-to-moderate postpartum depression: a double-blind, randomized clinical trial. Pharmacopsychiatry 50, 64
68. Kell, G., Rao, A., Beccaria, G., Clayton, P., Inarejos-Garcı´a, A.M., Prodanov, M., 2017. affrons a novel saffron extract (Crocus sativus L.) improves mood in healthy adults over 4 weeks in a double-blind, parallel, randomized, placebo-controlled clinical trial. Complement. Ther. Med. 33, 58
64. Khalili, M., Hamzeh, F., 2010. Effects of active constituents of Crocus sativus L., crocin on streptozocin-induced model of sporadic Alzheimer’s disease in male rats. Iran Biomed. J 14, 59
65. Khayatnouri, M., Safavi, S., Safarmashaei, S., Babazadeh, D., Mikailpourardabili, B., 2011. The effect of saffron orally administration on spermatogenesis index in rat. Adv. Environ. Biol. 5, 1514
1521. Khazdair, M.R., Boskabady, M.H., Hosseini, M., Rezaee, R.M., Tsatsakis, A., 2015. The effects of Crocus sativus (saffron) and its constituents on nervous system: a review. Avicenna J. Phytomed. 5, 376
391. Khorasany, A.R., Hosseinzadeh, H., 2016. Therapeutic effects of saffron (Crocus sativus L.) in digestive disorders: a review. Iran J. Basic Med. Sci. 19, 455
469. Kianbakht, S., Ghazavi, A., 2011. Immunomodulatory effects of saffron: a randomized double-blind placebo-controlled clinical trial. Phytother. Res. 25, 1801
1805. Lahmass, I., Sabouni, A., Elyoubi, M., Benabbes, R., Mokhtari, S., Saalaoui, E., 2017. Anti-diabetic effect of aqueous extract Crocus sativus L. in tartrazine induced diabetic male rats. Physiol. Pharmacol. 21, 312
321. Lari, P., Abnous, K., Imenshahidi, M., Rashedinia, M., Razavi, M., Hosseinzadeh, H., 2013. Evaluation of diazinon-induced hepatotoxicity and protective effects of crocin. Toxicol. Indust. Health 31, 367
376. Lopresti, A.L., Drummond, P.D., Inarejos-Garcı´a, A.M., Prodanov, M., 2018. affrons, a standardised extract from saffron (Crocus sativus L.) for the treatment of youth anxiety and depressive symptoms: a randomised, double-blind, placebo-controlled study. J. Affect. Disord. 232, 349
357. Mansoori, P., Akhondzadeh, S., Raisi, F., Ghaeli, P., Jamshidi, A., Nasehi, A., 2011. A randomized, double-blind, placebo-controlled study of safety of the adjunctive saffron on sexual dysfunction induced by a selective serotonin reuptake inhibitor. J. Med. Plants 1, 121
130. Martin, G., Goh, E., Neff, A., 2002. Evaluation of the developmental toxicity of crocetin on Xenopus. Food Chem. Toxicol. 40, 959
964. Mazidi, M., Shemshian, M., Mousavi, S.H., Norouzy, A., Kermani, T., Moghiman, T., et al., 2016. A double-blind, randomized and placebocontrolled trial of saffron (Crocus sativus L.) in the treatment of anxiety and depression. J. Complement. Integr. Med. 13, 195
199. Mehri, S., Abnous, K., Khooei, A., Mousavi, S.H., Shariaty, V.M., Hosseinzadeh, H., 2015. Crocin reduced acrylamide-induced neurotoxicity in Wistar rat through inhibition of oxidative stress. Iran J. Basic Med. Sci. 18, 902
908. Moallem, S.A., Afshar, M., Etemad, L., Razavi, B.M., Hosseinzadeh, H., 2013. Evaluation of teratogenic effects of crocin and safranal, active ingredients of saffron, in mice. Toxicol. Ind. Health 32, 1
7. Modaghegh, M., Shahabian, M., Esmaeili, H., Rajbai, O., Hosseinzadeh, H., 2008. Safety evaluation of saffron (Crocus sativus) tablets in healthy volunteers. Phytomedicine 15, 1032
1037. Mohajeri, D., Mousavi, G., Mesgari, M., Doustar, Y., Khayat Nouri, M., 2007. Subacute toxicity of Crocus sativus L.(saffron) stigma ethanolic extract in rats. Am. J. Pharmacol. Toxicol. 2, 189
193.

530

SECTION | VI Saffron and health

Mohamadpour, A.H., Ayati, Z., Parizadeh, M.R., Rajbai, O., Hosseinzadeh, H., 2013. Safety evaluation of crocin (a constituent of saffron) tablets in healthy volunteers. Iran J. Basic Med. Sci. 16, 39
46. Mohammadzadeh, L., Hosseinzadeh, H., Abnous, K., Razavi, B.M., 2018. Neuroprotective potential of crocin against malathion-induced motor deficit and neurochemical alterations in rats. Environ. Sci. Pollut. Res. Int. 25, 4904
4914. Mollazadeh, H., Emami, S.A., Hosseinzadeh, H., 2015. Razi’s Al-Hawi and saffron (Crocus sativus): a review. Iran J. Basic Med. Sci. 18, 1153
1166. Moshiri, E., Basti, A.A., Noorbala, A.A., Jamshidi, A.H., Hesameddin Abbasi, S., Akhondzadeh, S., 2006. Crocus sativus L. (petal) in the treatment of mild-to-moderate depression: a double-blind, randomized and placebo-controlled trial. Phytomedicine 13, 607
611. Mousavi, B., Bathaie, S.Z., Fadai, F., Ashtari, Z., Ali Beigi, N., Farhang, S., et al., 2015. Safety evaluation of saffron stigma (Crocus sativus L.) aqueous extract and crocin in patients with schizophrenia. Avicenna J. Phytomed. 5, 413
419. Noorbala, A.A., Akhondzadeh, S., Tahmacebi-Pour, N., Jamshidi, A.H., 2005. Hydro-alcoholic extract of Crocus sativus L. versus fluoxetine in the treatment of mild-to-moderate depression: a double-blind, randomized pilot trial. J. Ethnopharmacol. 97, 281
284. Ozaki, A., Kitano, M., Furusawa, N., Yamaguchi, H., Kuroda, K., Endo, G., 2002. Genotoxicity of gardenia yellow and its components. Food Chem. Toxicol. 40, 1603
1610. Ramadan, A., Soliman, G., Mahmoud, S.S., Nofal, S.M., Abdel-Rahman, R.F., 2012. Evaluation of the safety and antioxidant activities of Crocus sativus and Propolis ethanolic extracts. J. Saudi. Chem. Soc. 16, 13
21. Razavi, B.M., Hosseinzadeh, H., 2015. Saffron as an antidote or a protective agent against natural or chemical toxicities. Daru. 23, 31. Available from: https://doi.org/10.1186/s40199-015-0112-y. Razavi, B.M., Hosseinzadeh, H., Movassaghi, A.R., Imenshahidi, M., Abnous, K., 2013a. Protective effect of crocin on diazinon induced cardiotoxicity in rats in subchronic exposure. Chem. Bio. Interact. 203, 547
555. Razavi, M., Hosseinzadeh, H., Abnous, K., Motamedshariaty, V.S., Imenshahidi, M., 2013b. Crocin restores hypotensive effect of subchronic administration of diazinon in rats. Iran J. Basic Med. Sci. 16, 64
72. Riahi-Zanjani, B., Balali-Mood, M., Mohammadi, E., Badie-Bostan, H., Memar, B., Karimi, G., 2015. Safranal as a safe compound to mice immune system. Avicenna J. Phytomed. 5, 441
449. Sadi, R., Mohammad-Alizadeh-Charandabi, S., Mirghafourvand, M., Javadzadeh, Y., Ahmadi-Bonabi, A., 2016. Effect of saffron (Fan Hong Hua) on the readiness of the uterine cervix in term pregnancy: a placebo-controlled randomized trial. Iran Red. Crescent. Med. J. 18, e27241. Available from: https://doi.org/10.5812/ircmj.27241. Saks, M., Upreti, S., Dang, R., 2017. Genotoxicity: mechanisms, testing guidelines and methods. Global J. Pharm. Pharmaceutical. Sci. 1, 1
6. Schmidt, M., Betti, G., Hensel, A., 2007. Saffron in phytotherapy: pharmacology and clinical uses. Wien. Med. Wochenschr. 157, 315
319. Sepahi, S., Mohajeri, S.A., Hosseini, S.M., Khodaverdi, E., Shoeibi, N., Namdari, M., et al., 2018. Effects of crocin on diabetic maculopathy: a placebo-controlled randomized clinical trial. Am. J. Ophthalmol. 190, 89
98. Shafiee, M., Arekhi, S., Omranzadeh, A., Sahebkar, A., 2017. Saffron in the treatment of depression, anxiety and other mental disorders: current evidence and potential mechanisms of action. J. Affect. Disord. 227, 330
337. Shahi, T., Assadpour, E., Jafari, S.M., 2016. Main chemical compounds and pharmacological activities of stigmas and tepals of ‘red gold’; saffron. Trends Food Sci. Technol. 58, 69
78. Shahmansouri, N., Farokhnia, M., Abbasi, S.H., Kassaian, S.E., Noorbala Tafti, A.A., Gougol, A., et al., 2014. A randomized, double-blind, clinical trial comparing the efficacy and safety of Crocus sativus L. with fluoxetine for improving mild-to-moderate depression in post percutaneous coronary intervention patients. J. Affect. Disord. 155, 216
222. Tabeshpour, J., Sobhani, F., Sadjadi, S.A., Hosseinzadeh, H., Mohajeri, S.A., Rajabi, O., et al., 2017. A double-blind, randomized, placebo-controlled trial of saffron stigma (Crocus sativus L.) in mothers suffering from mild-to-moderate postpartum depression. Phytomedicine 36, 145
152. Taheri, F., Bathaie, S.Z., Ashrafi, M., Ghasemi, E., 2014. Assessment of crocin toxicity on the rat liver. Modares. J. Med. Sci. Pathobiology 17, 67
79 (in Persain). Vahdati-Hassani, F., Naseri, V., Razavi, B.M., Mehri, S., Abnous, K., Hosseinzadeh, H., 2014. Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus. Daru. 22, 16. Available from: https://doi.org/10.1186/2008-2231-22-16. Vahdati-Hassani, F., Mehri, S., Abnous, K., Birner-Gruenberger, R., Hosseinzadeh, H., 2017. Protective effect of crocin on BPA-induced liver toxicity in rats through inhibition of oxidative stress and downregulation of MAPK and MAPKAP signaling pathway and miRNA-122 expression. Food Chem. Toxicol. 107, 395
405. Yamashita, S.I., Kakinuma, T., Umigai, N., Takara, T., 2018a. Safety evaluation of excessive intake of crocetin in healthy adult volunteers: a randomized, double-blind, placebo-controlled, parallel-group comparison trial. Jpn. Pharmacol. Ther. 46, 393
401. Yamashita, S.I., Kakinuma, T., Umigai, N., Takara, T., 2018b. Safety evaluation of long-term intake of crocetin in healthy adult volunteers: a randomized, double-blind, placebo-controlled, parallel-group comparison trial. Jpn. Pharmacol. Ther. 46, 801
809. Yang, X., Chen, X., Fu, Y., Luo, Q., Du, L., Qiu, H., et al., 2018. Comparative efficacy and safety of Crocus sativus L. for treating mild-to-moderate major depressive disorder in adults: a meta-analysis of randomized controlled trials. Neuropsychiatr. Dis. Treat. 14, 1297
1305. Yousefsani, B.S., Mehri, S., Pourahmad, J., Hosseinzadeh, H., 2018. Crocin prevents sub-cellular organelle damage, proteolysis and apoptosis in rat hepatocytes: a justification for its hepatoprotection. Iran. J. Pharm. Res. 17, 553
562. Zeynali, F., Dasthi, M., Anvari, M., Hosseini, S., Miresmaeili, S., 2009. Studing teratogenic and abortification effects of different doses of saffron (Crocus sativus) decoction in whole gestational period and the 3rd trimester of gestaional peroid in mice. Inte. J. Reprod. Biomedi. 7.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A 2AA. See 2-Amino-antracene (2AA) Abiotic stresses, molecular response to, 255 Absorbing roots, 94f, 96
97 Acanthopanax (Kalopanax septemlobus), 499 Acari, 177
182 Acaulospora morrowiae, 86 Accessions of saffron, 220t Acclimation of saffron, 232 Acetylcholineesterase inhibitors (AChEIs), 446 Acidification potential (AP), 369, 370t Acne mask, 495 Aconitum napellus. See White monkshood (Aconitum napellus) Activation of kinase (Akt1), 407
408 Actual crop evapotranspiration (ETa), 150 ACZs. See Agroclimatic zones (ACZs) AD. See Alzheimer’s disease (AD) ADAS-cog. See Alzheimer’s disease assessment scale cognitive subscale (ADAS-cog) Adiponectin, 455 Adulteration, 224
226, 321 detection, 321
332 chromatographic techniques, 322
325 e-nose, 331
332 1 H NMR, 328 IR spectroscopy, 325
328 molecular techniques, 328
331 forms in saffron and saffron powder, 322t Advanced glycation end products (AGEs), 454, 473 Adventitious shoots, 233
235 AEP. See Aquatic eutrophication potential (AEP) Aeroponics, 207
208 Afghanistan, saffron production in, 342
343 Aflatoxin B1 (AFB1), 489 Aflatoxins, 309 Aflatoxin-producing fungi, 308 AFLP analysis, 226 aflR1 gene, 309 Ag II. See Angiotensin II (Ag II) AGAMOUS1-like genes (CsAG1), 247
248 AGEs. See Advanced glycation end products (AGEs) Aggregated environmental indicator (EcoX), 371
372, 371t Agriculture, 381 ecosystem services and impacts from, 382

Agroclimatic zones (ACZs), 162 Agroecological zoning, 162
163 Agronomical practices of saffron corm corm lifting time and storage, 101
102 corm size classification, 106
107 corm size/weight, 104
106 hormones application, 112 planting beds application of organic and chemical fertilizers, 107
108 crop residue, 108
109 intercropping, 109
110 planting under controlled environment, 110
112 planting depth, 103
104 planting time, 103 row spacing, corm density, 104 AGS cells. See Gastric adenocarcinoma cells (AGS cells) AHP. See Analytical hierarchy process (AHP) Airway smooth muscle mechanisms, 463
464 relaxant effect of saffron derivatives, 462
463 of saffron extracts, 462 AKIS Network. See Smart Agricultural Knowledge and Innovation Systems Network (AKIS Network) Akt1. See Activation of kinase (Akt1) Alanine aminotransferase (ALT), 479, 518 Alcoholic and nonalcoholic beverages, 282
283 Aldehyde dehydrogenase (ALDH), 252 ALDH. See Aldehyde dehydrogenase (ALDH) Alisma orientalis. See Rhizoma alismatis (Alisma orientalis) Alkaline phosphatase (ALP), 479 Alkanna tinctoria. See Dyer’s alkanet (Alkanna tinctoria) “All Red” saffron. See Sargol saffron All-trans retinoic acid (ATRA), 520 Allele-specific PCR (AS-PCR), 329 Allium cepa. See Onion (Allium cepa) Allium sativa. See Garlic (Allium sativa) Aloe whitening cream, 495 ALP. See Alkaline phosphatase (ALP) Alpha 2 adrenergic receptor blocking agents, 433 α-crocin. See Crocin 1 ALT. See Alanine aminotransferase (ALT)

Aluminum foil, 302 Aluminum neurotoxicity, 426 Alzheimer’s disease (AD), 283, 426, 445
446 epidemiology, 445 herbal medicines and, 449 pathogenesis, 445
446 pharmacological therapy, 446 saffron in, 446
448 clinical trials, 448 effects on CNS, 446
448 neuroprotective activity of saffron, 448 signs and symptoms, 445 Alzheimer’s disease assessment scale cognitive subscale (ADAS-cog), 427, 445 Ames/Salmonella test system, 521 AMF. See Arbuscular mycorrhizal fungi (AMF) Amine reuptake inhibitors, 433 2-Amino-antracene (2AA), 521 AMOVA. See Analysis of molecular variance (AMOVA) AMP-activated protein kinase pathways, 473
476 Amyloid-β peptide (Aβ peptide), 447
448 Analysis of molecular variance (AMOVA), 224t Analytical hierarchy process (AHP), 162 Ancient Iran, saffron uses in, 25
28 cosmetic use, 26 medical and pharmaceutical uses, 25 ritual uses, 26
28 burning and incensing saffron, 26 decoration of steeds with saffron, 27 donating and gifting saffron, 26
27 drinking saffron syrup, 27 magical uses, 27 saffron applications in astronomy and astrology, 27
28 writing uses, 27 saffron for food applications, 25 Angelica pubescens. See Pubescent angelica (Angelica pubescens) Angelica sinensis, 496, 500 Angiitis, Chinese medicine for treatment of, 496 Angiogenesis, 486 Angiotensin II (Ag II), 453 Animal manures, 64 Ankylosing spondylitis, medicine for treating, 503

531

532

Index

ANNs. See Artificial neural networks (ANNs) Annual summer weeds, 173 Annual winter weeds, 173 Anthocyanins, 249 Antiacne ointment, 497 Antianxiety drugs, 433 effects, 438 Antiarrhythmic effects of saffron, 452
453 Antiatherosclerotic effects of saffron and active constituents, 454
455 Antibacterial packaging effect on dried stigma microbiological quality, 315
316 Anticancer properties of saffron breast cancer, 487
488 cervical cancer, 489
490 colorectal cancer, 487 gastric cancer, 490
491 hepatic cancer, 489 leukemia, 488 lung cancer, 486
487 pancreatic cancer, 487 prostate cancer, 490 skin cancer, 490 Anticarcinogenic effect of saffron, 403, 488 Antidepressants, 433
435 and antianxiety drugs, 433 mechanism of action, 434 pharmacokinetics, 434 properties of saffron, 437
438 side effects, 434
435 Antidepression, 435 Antihyperglycemic effects of saffron, 473 mechanisms of saffron, 473
476 Antiinflammatory effects of saffron and derivatives, 406
412, 413t derivatives, 409
412 extracts, 406
407, 407t petals, 407
409 Antiischemic effects of saffron, 452
453 Antimicrobial composition, 498 Antimicrobial packaging, 304 Antioxidants, 211
212 Anxiety, 284, 432
433 causes, 433 epidemiology, 432
433 pathophysiology, 432 traditional medicine for treatment of, 435 in vivo studies on effects of saffron and active compounds, 436
437 Anxiolytic properties of saffron, 437
438 AP. See Acidification potential (AP) Apocarotenoids, 274, 277t, 278
279, 283 biosynthesis, 251
252 Apoptosis, 486 Apparent dormancy, 95 Applications of saffron, 282
285 in astrology, 27
28 in astronomy, 27
28 cosmetics, 284
285 food industry, 282
283 in Iranian daily life, 30
32 dyeing, 30
31

food, 31
32 odor and aroma, 30 pharmaceutical industry, 282
285, 436 AquaCrop model, 78, 151
152 Aquatic eutrophication potential (AEP), 369
370, 370t Aqueous two-phase system (ATPS), 267t Aquilaria sinensis. See Chinese eaglewood (Aquilaria sinensis) Arabic medicine, 394 Arbuscular mycorrhizal fungi (AMF), 255 Arctium tomentosum. See Burdock (Arctium tomentosum) Arillus longan (Euphoria longan), 500 Aroma effects of, 296
297 saffron use as, 30 Arthritis, 497 Artificial drying methods, 292
293, 311 Artificial neural networks (ANNs), 143
146, 148
149 application for saffron, 144
146 AS-PCR. See Allele-specific PCR (AS-PCR) Asarum heterotropoides, 500 Asarum sieboldii, 500 Ascites due to cirrhosis, traditional Chinese medicine composition for treating, 503 Aspartate aminotransferase (AST), 479, 518 Aspergillus spp., 308 A. flavus, 309 A. niger, 102 A. parasiticus, 309 Assimilation efficiency, 346 AST. See Aspartate aminotransferase (AST) Astrology, saffron applications in, 27
28 Astronomy, saffron applications in, 27
28 Atherogenic index, 455 Atherosclerosis index, 455 ATPS. See Aqueous two-phase system (ATPS) ATR-FTIR spectroscopy. See Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR spectroscopy) ATRA. See All-trans retinoic acid (ATRA) Atractylodes macrocephala. See Baizhu (Atractylodes macrocephala) Atrioventricular nodes (AV nodes), 452 Attapulgite-saffron suntan lotion, 496 Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR spectroscopy), 328 Automatic planting machines, 192
194 fully automated seven-row saffron corm planter, 194f two-row saffron corm planter, 194f Auxin, 112, 233
234 AV nodes. See Atrioventricular nodes (AV nodes) Axial points, 147 Ayat al-korsi, 6
7 Ayurveda saffron, 16

B Babiana, 43

Bacillus amyloliquefaciens, 255 Bacillus cereus, 308 Bacillus croci, 183 Bacillus subtilis, 255 BAECs. See Bovine aortic endothelial cells (BAECs) Baikal skullcap root (Scutellaria baicalensis), 498 Baizhu (Atractylodes macrocephala), 498, 500 Balassa index. See Revealed comparative advantage (RCA) BALF. See Bronchoalveolar lavage fluid (BALF) Balgham, 395 Bank of Plant Germplasm of Cuenca (BGVCU), 220 Bar-MCA. See Barcoding melting curve analysis method (Bar-MCA) Barcoding melting curve analysis method (BarMCA), 329 Bath cream, 494 Bax protein, 487
488 BCH. See β-carotene hydroxylase (BCH) Bchi gene, 44
45 BDNF. See Brain-derived neurotrophic factor (BDNF) Bed preparation for corm planting, 189
190 Beneficial water-related approaches for saffron production, 85
87 Benzo[a]-pyrene (BP), 521 Beta function, 157 Beta-amyloid aggregation, 426 β-carotene, 250 β-carotene hydroxylase (BCH), 250, 253 β-hexosaminidase, 408 β-LCY. See Lycopene β-cyclase (β-LCY) BGV-CU. See Bank of Plant Germplasm of Cuenca (BGV-CU) Bioactive ingredients/components/compounds applications, 282
285 cosmetics, 284
285 food industry, 282
283 pharmaceutical industry, 282
285 chromatographic systems and analytical conditions, 275t classification according to National Standards of Iran, 263t of dried saffron stigmas, 265f e-nose technique, 279
281 extraction of saffron, 264
272, 267t DLLME, 272 HD, 270 HPE, 271 maceration, 266
270 microextraction methods, 271 SBSE, 271
272 Soxhlet extraction, 270 SPME, 271 studies dealing with extraction, 267t supercritical fluid extraction, 270
271 UF, 271 GC-MS, 279, 280t gradient elution system for saffron analysis, 274t

Index

HPLC, 273
279 international classification of saffron stigmas, 262 Iranian trade categories, 262
264 physicochemical classification of saffron, 262t UV-Vis spectrophotometry, 272
273, 277t Biodegradable films, 304 Biodegradation, 264 Biodiversity, 384 Biogenetic resources, 384 Biological cosmetic cream, 494 Biomass, 151
152 partitioning, 122, 133, 170 Biopolymers, 304 Biotic organisms, 169 Black pepper (Piper nigrum L.), 314 Blood pressure (BP), 453 effects of saffron and active constituents on, 453
454 Blood stasis, traditional Chinese medicine preparation for treating, 502 Body mass index (BMI), 471 Bone injury, drug for treating, 504 Botany of saffron contractile roots, 40
41 corm, 41 Crocus, 38
40 Iridaceae, 41
43 and phylogeny, 43
46 Bovine aortic endothelial cells (BAECs), 454 BP. See Benzo[a]-pyrene (BP); Blood pressure (BP) Brain apoplexy, Chinese medicine for treating, 500 Brain-derived neurotrophic factor (BDNF), 436 growth factor, 432 Brand, 358 Brassica rapa subsp. rapa. See Turnip (Brassica rapa subsp. rapa) Breaking soil crust, 174 Breast cancer, 487
488 topical treatment for, 500 Broad-leaved herbicides, 176 Bronchoalveolar lavage fluid (BALF), 407
408 Bronchodilatory effect of saffron and derivatives, 462
464 relaxant effect mechanisms of airway smooth muscle, 463
464 of saffron derivatives on airway smooth muscle, 462
463 of saffron extracts on airway smooth muscle, 462 Buddhism, saffron color in, 19 “Bunch”. See Daste of saffron Bupleurum chinense, 500 Bupleurum scorzonerifolium, 500 Burdock (Arctium tomentosum), 499 Burkholderia gladioli, 183 Burreed tuber (Sparganium stoloniferum), 500

C C-C chemokine receptor type 3 (CCR3), 408 C-X-C chemokine receptor type 2 (CXCR2), 408 C10H14O. See Safranal (SFR) CaCCD2 isoform, 251
252 Caesalpinia sappan, 500 Calibration model, 150 Callus and cell culture, 232
233 cAMP response element binding protein (CREB), 436 Cancer, defined, 485 Carbon dioxide (CO2), 368
369 Carbon footprint, 380
381 Carbon sequestration, 383 Cardaria draba, 174 Cardiac hypertrophy, protective effects against, 453 Cardinal temperatures, 155 Cardiovascular complications of diabetes, 478 pharmacological effects of saffron, 452
457 antiarrhythmic and antiischemic effects, 452
453 antiatherosclerotic effects of saffron and active constituents, 454
455 effects of saffron and active constituents on blood pressure, 453
454 protective effects against cardiac hypertrophy, 453 protective effects and active constituents on natural and chemical toxins, 455
457 traditional Chinese medicine for treating cardiovascular diseases, 504 Carotenoid cleavage dioxygenases (CCDs), 251 Carotenoid isomerase (CRTISO), 250 Carotenoids, 249
251, 414 biosynthesis genetic regulation, 253
254 in plants, 249
250 in saffron, 251 Carthamus lanatus, 23
24 Carthamus tinctorios, 19
20 Cassia twig (Cinnamomum cassia), 496 Castile
La Mancha, 221, 292 Cataphylls, 47
49 Cataracts, Chinese medicine for treating, 501 CCDs. See Carotenoid cleavage dioxygenases (CCDs); Central composite designs (CCDs) CCR3. See C-C chemokine receptor type 3 (CCR3) cDNA encoding, 255 PSY and PDS, 251 CDR-SOB. See Clinical dementia rating scale sums of boxes (CDR-SOB) Cell culture, Callus and, 232
233 CEN/TFL1-like gene. See CENTRORADIALIS/ TERMINAL FLOWER1-like gene (CEN/ TFL1-like gene) Center point designs, 147 Central composite designs (CCDs), 146
147, 255

533

CCD7, 252 Central nervous system (CNS), 410, 423, 431
432, 446 effects of saffron on, 446
448 CENTRORADIALIS/TERMINAL FLOWER1like gene (CEN/TFL1-like gene), 247
248 Cereal products, 282 Cerebrovascular diseases, traditional Chinese medicine for treating, 504 Cervical cancer, 489
490 Cervical spondylosis, Traditional Chinese medicine capable of treating, 501
502 CGR. See Crop growth rate (CGR) Chang Mal, 6 Charm, Saffron in, 6
7 Chaveris, 23
24 Chemical analysis of saffron stigmas, 405 Chemical control in weed management, 175
176 Chemical fertilizer, 368 in saffron cultivation, 107
108 Chemical toxins, 455
457 Chemoprevention, 485 Chenopodium album, 171
173 Chinese eaglewood (Aquilaria sinensis), 498 Chinese medicine for angiitis treatment, 496 for brain apoplexy treatment, 500 for cataracts treatment, 501 for gynecologic disease treatment, 500 for lung tumor treatment, 502 medicinal composition, 498 for osteoproliferation and herniated disk treatment, 502 for peptic ulcer treatment, 503 for rheumatic arthritis treatment and prevention, 499 Chinese yam (Dioscorea polystachya), 499 Chonspora tanella, 171
173 Chromatographic techniques, 322
325 Chronic cerebral hypoperfusion, 425 Chronic obstructive pulmonary disease, medicine for treating, 499 Chtonobdella limbata. See Hungry leeches (Chtonobdella limbata) CIF prices. See Cost, insurance, and freight prices (CIF prices) Cinnamomum cassia. See Cassia twig (Cinnamomum cassia) Cinnamomum verum. See Cinnamon (Cinnamomum verum) Cinnamon (Cinnamomum verum), 498 CK MB. See Creatine kinase MB (CK MB) Cladistics analysis, 43 Classical culturing method, 308
309 Climate change, 80
81 corm and, 112
113 impacts, 140 saffron response simulation effects on DR, 159
160 effects on flowering period length, 160
161

534

Index

Climate change (Continued) general impacts of global warming, 158
159 Climatic factors for crop production, 120
122 precipitation, 121
122 saffron cultivated areas in world and environmental criteria, 121t temperature, 120
121 Clinical dementia rating scale sums of boxes (CDR-SOB), 427, 445 Clinical studies of saffron, 427 Clostridium perfringens, 308 CNS. See Central nervous system (CNS) Cobb
Douglas production function, 344 Coefficient of determination (R2), 142 Coefficients of regression equations, 150 Cognitive impairments, 425 Cognitive theories, 433 Cold and flu symptomatic relief composition, 498 Cold plasma process effect on dried stigma microbiological quality, 313 Colorectal cancer, 487 Commodity, saffron as, 28
30 Compositions containing enriched natural crocin and/or crocetin, 496 Computer vision (CVS), 331
332 Contamination of saffron, 230, 307 Contractile roots, 40
41, 96
97 Conventional propagation methods, 230 Convoying, 398 Convoys, 398
402 Corm(s), 133 agronomical practices, 101
112 botanical criteria developmental stages and phonological description, 98 field age effect on corm production, 101 main and lateral buds, 94
96 mother and daughter corms, 93
94 nutrient content, 98
100 root system, 96
98 corm and climate change, 112
113 Crocus, 41, 42f, 52f digging, 188
189 planting automatic machines, 192
194 patterns, 191
192 traditional methods, 192 size, 59
60, 62
63 sorting, 189 saffron corm sorter, 190f Cormlets, 229
232, 235
236, 237f Correlation analysis, 145
146 Correlation coefficient (R), 142 Cosmetics, saffron in, 284
285 Cost, insurance, and freight prices (CIF prices), 350 “Coupe´” saffron. See Sargol saffron Cover crops, 175 COX-2. See Cyclooxygenase-2 (COX-2) CPWC. See Critical period for weed control (CPWC) Creams, 494
495

Creatine kinase MB (CK MB), 452
453 CREB. See cAMP response element binding protein (CREB) Crepis saneta, 171
173 Critical period for weed control (CPWC), 171 Crocetin, 252
253, 411, 446
447, 453, 461, 485
486, 489, 520 di-glucose ester, 424 gentiobiose glucose ester, 424 immunomodulatory effects, 415
417 sugar esters, 275t, 278
279, 283 vasomodulatory effects of crocetin in hypertension, 453
454 Crocin, 252
253, 409
411, 435, 447, 455, 461, 471
473, 485
486, 488, 518
519, 521 clinical trials on antidepressant and anxiolytic effect, 439t effectiveness of, 424 experimental studies related to safety and toxicity, 523t immunomodulatory effects, 415 intraperitoneal injection, 437 mechanistic pathways for antidepressant effects, 440f single posttraining injection, 424 Crocin 1, 410
411, 485
486 Crocus, 3, 38
40 C. almehensis, 41, 42f C. angustifolius, 41 C. biflorus, 41, 42f C. cartrightianus, 38 C. cartwrightianus, 41, 42f, 219 C. caspius, 41, 42f C. flavus, 41, 42f C. gilanicus, 41 C. hadriaticus, 41, 42f, 54, 55f C. hermoneus, 41, 42f C. korolkowii, 41 C. michelsonii, 41 C. oreocreticus, 53
54, 54f C. pallasii, 41, 42f C. sativus L., 291, 397, 500, 525 C. sieberi, 41, 42f C. speciosus, 41 C. thomasii, 41, 42f, 54, 55f C. vernus, 40f Crocus cartwrightianus. See Wild saffron (Crocus cartwrightianus) Crocus palasii. See Joe ghasem saffron (Crocus palasii) Crocus sativus L. See Saffron (Crocus sativus L.) “CROCUSBANK” project, 220 Crop coefficients, 67
70, 69f growth, 153 models, 78, 139 protection of saffron pathogens, 182
183 pests, 176
182 weeds, 169
176 residue, 108
109 simulation models, 148, 152

yields, 28, 140, 148
149 Crop evapotranspiration (ETc), 150 Crop growth rate (CGR), 126, 126t Crop-specific parameters of saffron, 154 Crop-weather models for saffron yield prediction, 140
142 CRTISO. See Carotenoid isomerase (CRTISO) Crust breaking, 72, 190
191 CsAG1. See AGAMOUS1-like genes (CsAG1) CsatCEN/TFL1-like gene, 247
248 CsBCH1 transcripts, 253 CsCCD2 isoform, 251
252 CsCCD2-t isoform, 251
252 CsCCD2b isoform, 251
252 CsGT. See Stress-inducible glycosyltransferase (CsGT) CsLcyB2a, 253 CsPR10 encoding, 255 CsSAP09 transcription factor, 254 CsUGT2 gene, 252
253 CsUGT3 gene, 252
253 Cube points. See 2K factorial points Cultivated saffron, 47
51 Cultivation under controlled environments, 86
87 Cultural function, 381 Cumin (Cuminum cyminum), 109, 498 Cup-shaped containers, 201 Curcuma longa. See Turmeric (Curcuma longa) Curcuma root, 500 Curcuma zedoaria. See Zedoary (Curcuma zedoaria) Cushing’s disease, 432 CVS. See Computer vision (CVS) CXCR2. See C-X-C chemokine receptor type 2 (CXCR2) Cyanogenic glucosides, 249 Cyclooxygenase-2 (COX-2), 408 Cyperus rotundus, 500 Cytokinin, 112

D DAFI. See Days after first irrigation (DAFI) Daily weather data, 150 Damp-heat stagnation, medicine for treating, 502
503 Daste of saffron, 200, 200f, 264 Daughter corms, 93
94 Days after first irrigation (DAFI), 150 Debij cakes (saffron cookies), 6 Degenerative eye disorders, 500 Dehydration, 291
292, 295 artificial drying methods, 292
293 drying methods, 291
292 effects of drying on color, aroma, and taste, 296
297 freeze drying, 295 hybrid photovoltaic
thermal solar dryer, 293
294 infrared thin-layer drying, 294 microwave drying, 295
296 of saffron stigmas, 292

Index

traditional methods, 292 Delayed type of hypersensitivity (DTH), 417 DEN. See Diethylnitrosamine (DEN) Deoxycorticosterone acetate (DOCA), 462 Deoxyxylulose-5-phosphate (DXP), 249
250 1-Deoxyxylulose-5-phosphate reductoisomerase (DXR), 249
250 Depression, 284, 432
433 causes, 433 epidemiology, 432
433 pathophysiology, 432 traditional medicine for treatment, 435 in vivo studies on effects of saffron and active compounds, 436
437 Dermatitis, ointment for treating, 497 Descurainia sophia. See Tinglizi (Descurainia sophia) Desserts, 282 Development rate (DR), 155 climate change effects, 159
160 Developmental responses in crop species, 155 Diabetes, saffron effects on, 472
479 antihyperglycemic effects of saffron, 473 antihyperglycemic mechanisms, 473
476 complications, 476
479 experimental studies, 474t Diabetic nephropathy, 477
478 Diabetic neuropathy, 476
479 Diethylnitrosamine (DEN), 489 Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), 328 Dimethylallyl diphosphate (DMAPP), 249 Dimethylbenz[a] anthracene (DMBA), 490 Dioscorea polystachya. See Chinese yam (Dioscorea polystachya) Diospyros ebenum. See Ebony (Diospyros ebenum) Direct energy, 372 Direct shoots, 234, 234f Direct
indirect energy ratio, 373
374 Dispersive liquid
liquid microextraction (DLLME), 272 Divaricate saposhnikovia (Saposhnikovia divaricate), 499 DLLME. See Dispersive liquid
liquid microextraction (DLLME) DM. See Dry matter (DM) DMAPP. See Dimethylallyl diphosphate (DMAPP) DMBA. See Dimethylbenz[a] anthracene (DMBA) DNA markers, 328
329 DNA methylation, 219
220 DNA-based traceability, 224
226 Do-bayti, Saffron in, 9
10 DOCA. See Deoxycorticosterone acetate (DOCA) Domestic Resource Cost (DRC), 351
353 Dominant weeds in saffron fields, 171
173 annual summer weeds, 173 annual winter weeds, 173 perennials, 173 Donepezil, 446, 448 Dormancy, 95

DR. See Development rate (DR) DRC. See Domestic Resource Cost (DRC) Dried stigma, microbiological quality of antibacterial packaging effect on, 315
316 cold plasma process effect on, 313 drying processes effect on, 311
312 hurdle technology effect on, 316
317 irradiation treatment effect on, 313
314 ozone treatment effect on, 313 DRIFTS. See Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) Drought stress, 84
86, 100 Drugs for kidney health, 496 Dry matter (DM), 153 production, 153
154 Drying effects on color, 296
297 methods, 291
292 processes effect on microbiological quality of dried stigma, 311
312 artificial drying, 311 freeze-drying process, 311
312 traditional drying, 311 DTH. See Delayed type of hypersensitivity (DTH) DXP. See Deoxyxylulose-5-phosphate (DXP) DXR. See 1-Deoxyxylulose-5-phosphate reductoisomerase (DXR) Dyeing and saffron, 30
31 Dyer’s alkanet (Alkanna tinctoria), 498 Dynamic simulation models, 148
154 advances in simulation models development for saffron, 150
152 radiation-based model for saffron growth, 152
154 Dynamic water-based model, 150 Dysmenorrhea, traditional Chinese composition for treating, 501

E e-commerce. See Electronic commerce (ecommerce) e-nose. See Electronic nose (e-nose) E-selectin (E-sel), 408 EAE. See Experimental autoimmune encephalomyelitis (EAE) Ebony (Diospyros ebenum), 495 EC. See Electrical conductivity (EC) Ecological economic analysis of energy use, 372
380 energy economic indicators, 378
380 of energy input, 375
378 Ecological energy indicators, 373
374 Economic advantages of saffron production, 353 Economic comparative advantage, 349
353 Domestic Resource Cost (DRC), 352
353 Effective Protection Coefficient (EPC), 352 Nominal Protection Coefficient (NPC), 352 policy analysis matrix, 349
351, 350t Revealed comparative advantage (RCA), 349 Economic efficiency, 348
349

535

of saffron cultivation, 348
349 Economic performance of saffron, 346 Ratio of return to cost (RRC), 346 Return on investment (ROI), 346 Economic productivity of saffron, 344
345 labor productivity of saffron, 345 land productivity of saffron, 345 water productivity of saffron, 345 Ecophysiology of saffron climatic factors for crop production, 120
122 environmental change effects on quality of saffron, 134
135 growth parameters, 123
134 lifecycle, 122
123 yield determination, 135 Ecosystem functions, 381
385 valuation, 382 Ecosystem services, 381
385 and impacts from agriculture, 382 Edible films, 304 Effective Protection Coefficient (EPC), 351
352 Efficiency, 346
349 ELECTRE method, 162 Electrical conductivity (EC), 82
83 Electron beam irradiation treatment, 314 Electronic commerce (e-commerce), 212
213 Electronic nose (e-nose), 279
281, 331
332 Electrospray ionization mass spectrometry (ESI-MS), 324 Ellobius fuscocapillus. See Southern mole vole (Ellobius fuscocapillus) Embryogenic calli, 238
239, 241 Embryogenic cells, 238 Empirical models of plant growth, 139 EMT. See Epithelial-mesenchymal transition (EMT) Emulsion liquid membrane, 267t Endocrine dysfunction, composition for treating, 501 Endoplasmic reticulum (ER), 410 Endothelial nitric oxide synthase (eNOS), 454 Endothelin-1 (ET-1), 406, 465, 467 Energy economic indicators, 378
380 economic productivity, 379
380 intensiveness, 379 output
input ratios, 373 productivity, 379 Energy use ecological economic analysis, 372
380 efficiency, 346
349, 378 of saffron cultivation, 348 eNOS. See Endothelial nitric oxide synthase (eNOS) Enterococci, 304 Environmental change effects on saffron quality, 134
135 Environmental economic analysis of saffron production carbon footprint, 380
381 ecological economic analysis of energy use, 372
380

536

Index

Environmental economic analysis of saffron production (Continued) ecosystem functions and services, 381
385 environmental impacts, 384
385 estimation of potential environmental impacts by LCA, 367
372 green policy analysis matrix of saffron, 385
386 Environmental pollution, 304 Environmental Protection Agency (EPA), 313 Eotaxin-1, 408 EPA. See Environmental Protection Agency (EPA) EPC. See Effective Protection Coefficient (EPC) Epigenetics stability, 222
224 Epilepsy, 425 Epithelial-mesenchymal transition (EMT), 407
408 9-cis-Epoxycarotenoid dioxygenases (NCEDs), 251 ER. See Endoplasmic reticulum (ER) ERK1/2. See Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) Erodium cicutarium, 171
173 Escherichia coli, 308
309 E. coli ATCC 29213, 309 ESI-MS. See Electrospray ionization mass spectrometry (ESI-MS) EST. See Expressed sequence tag (EST) ET-1. See Endothelin-1 (ET-1) ETCCDM. See Expert Team on Climate Change Detection and Monitoring (ETCCDM) Ethanolic extract of saffron, 423 Ethylene, 112 biosynthesis, 210 Eucommia (Eucommia ulmoides), 498 Eucommia ulmoides. See Eucommia (Eucommia ulmoides) Euphorbia helioscopia, 171
173 Euphoria longan. See Arillus longan (Euphoria longan) European Cooperation in Science and Technology COST Action FA1101, 225 Eutrophication, 369
370 EvaGreen real-time PCR approach, 329
331 Experimental autoimmune encephalomyelitis (EAE), 410 Expert Team on Climate Change Detection and Monitoring (ETCCDM), 140
141 Explant preparation of saffron, 230
231, 231f Exporters of saffron, 337
338 Exporting, 357
360 Expressed sequence tag (EST), 253
254 Externally applied Chinese medicine, 499 Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), 408 Extraction of saffron, 264
272, 267t dispersive liquid
liquid microextraction (DLLME), 272 high pressure extraction (HPE), 271 hydrodistillation (HD), 270 maceration, 266
270

microextraction methods, 271 solid-phase microextraction (SPME), 271 Soxhlet extraction, 270 stir-bar sorptive extraction (SBSE), 271
272 studies dealing with extraction, 267t supercritical fluid extraction, 270
271 ultrafiltration (UF), 271 Eyelid eczema, 504

F Face cream, 494 Fair-complexion face powder, 495 FAO-Penman-Monteith equation, 75 FAO
Penman method, 150 Farm age, 100 Farm yard manure (FYM), 174 Farsenyl diphosphate (FPP), 249
250 FDA. See US Food and Drug Administration (FDA) Feed additive, saffron as, 211
212 Ficus microcarpa. See Kowloon root (Ficus microcarpa) Field age effect on corm production, 101 Filago arvense, 171
173 First-order drugs, 396 Flooding, saffron response to, 87 Floral formula, 41
43 Flower development, 247
248 gene involvement, 248t emergence, 156
158 harvesting, 196f flowering rate of saffron fields, 196t harvesting device, 199f invented picker machines, 195
198 portable saffron harvesting device, 198f saffron harvesting with trolley, 197f traditional method, 194
195 induction, 120 initiation, 155
156 residues, 310 Flowering period length, climate change effects on, 160
161 Folk medicine, 8 Folklore, and saffron folkore in planting, growing, and harvesting, 4
5 and food, 5
6 in popular literature, 9
11 popular medicine, 8
9 in prayers, charms, and talismans, 6
7 uses of, 7
8 Food packaging, 301 production, 383
384 products, 504
512 healthy drink preparing from saffron pollen, 504
512 vegetable drinks, 504 saffron and, 5
6 saffron for food applications, 25, 31
32 saffron in food industry, 282
283 Forced flowering, 209

Formulations and patents of saffron, 494, 505t food products, 504
512 health care products, 496 skin care products, 494
496 therapeutic products, 496
504 Forsythia 3 intermedia. See Fructus forsythiae (Forsythia 3 intermedia) Fourier transform infrared spectroscopy (FTIR spectroscopy), 326
327 Fourth-order drugs, 396
397 FPP. See Farsenyl diphosphate (FPP) Freckle-whitening cream, 495 Freeze drying, 294f, 295, 295f, 311
312 Fructus forsythiae (Forsythia 3 intermedia), 499 FTIR spectroscopy. See Fourier transform infrared spectroscopy (FTIR spectroscopy) Fu Yang repair cream, 495 Fungal agents, 169 Fusarium oxysporum, 182 Fusarium rottings of saffron corm, 182 Fusarium solani, 182 FYM. See Farm yard manure (FYM)

G Galium tricorne, 171
173 Gametogenesis, 248 Gamma cell model, 316
317 Gamma irradiation treatment, 314 Gamma test, 145 Garlic (Allium sativa), 50
51 Gas chromatography-mass spectrometry (GCMS), 279, 280t Gastric adenocarcinoma cells (AGS cells), 490
491 Gastric cancer, 490
491 Gastric ulcer, traditional Chinese medicine for treating, 499
500 GBS. See Genotyping-by-sequencing (GBS) GC-MS. See Gas chromatography-mass spectrometry (GC-MS) Geissorhiza, 43 Genotyping-by-sequencing (GBS), 219 Gentiana lutea. See Large gentian root (Gentiana lutea) Geographic information systems (GISs), 162 GIS-based MCDM, 162 Geophyte plant, 93 Geostatistical techniques, 174 Geranyl diphosphate (GPP), 249
250 Geranylgeranyl diphosphate (GGPP), 249
250 Germplasms, 220 GGPP. See Geranylgeranyl diphosphate (GGPP) Gharabadin, 395 Ghashghah, 16 Ghavoot foufel, 8 Ghrelin, 472 “Ghyar”, 20 GIAHS. See Globally Important Agricultural Heritage Systems (GIAHS) Gibberellic acid, 112

Index

Gibberellin, 112 Gilan province, 6 Ginger (Zingiber officinale), 497 Ginkgo. See Ginkgo biloba leaf Ginkgo biloba leaf, 449 Ginseng. See Ginkgo biloba leaf GISs. See Geographic information systems (GISs) Gladiolus, 43 Glass, 302
303 Glechoma longituba, 500 Global warming, 112 impacts on saffron response, 158
159 Global warming potential (GWP), 368
369, 369t Globally Important Agricultural Heritage Systems (GIAHS), 206 Glomus coronatum, 86 Glucose, 473
476, 476f Glucosinolates, 249 Glutathione (GSH), 452, 489 Glutathione peroxidase (GPX), 452, 489, 519 “Golden condiment”, 219 Golden seal (Hydrastis canadensis), 498 Gonabad region, 206 Gout medicine, 498 GPP. See Geranyl diphosphate (GPP) GPX. See Glutathione peroxidase (GPX) Gradient elution system for saffron analysis, 274t Graminicides, 176 Greece, saffron production in, 341
342 Green policy analysis matrix of saffron, 385
386 Growth analysis index, 126
129 Growth of saffron, 119 Growth parameters of saffron, 123
134 CGR, 126 corms, 133 LAI, 125
126 LAR, 131 LWR, 131
133 NAR, 130 RGR, 126
130 source and sink relationship in growth organs, 134 whole plant, 133
134 GSH. See Glutathione (GSH) GSH-S-transferase (GST), 489 GST. See GSH-S-transferase (GST) GWP. See Global warming potential (GWP) Gynecologic diseases, Chinese medicine for treatment of, 500

H

1 H nuclear magnetic resonance (1H NMR), 328 HA. See Hemagglutination (HA) Habitat function, 381 Hadiths, 15
16, 21 Hair conditioner, 496 Hamilton Depression Scale (HAM-D scale), 438 Hand cream, 494

Hand weeding, 174 Hargreaves
Samani method, 150 Harmful rodents in saffron fields, 180
182 preventing and controlling rats in saffron fields, 181
182 short-tailed bandicoot rat, 180 Southern mole vole, 180 Harvest index (HI), 151
152 Harvesting monitoring of saffron flowers, 309
310 process of saffron, 307 saffron flowers, 196f flowering rate of saffron fields, 196t harvesting device, 199f invented picker machines, 195
198 portable saffron harvesting device, 198f saffron harvesting with trolley, 197f traditional method, 194
195 Hb. See Hemoglobin (Hb) HCT. See Hematocrit (HCT) HD. See Hydrodistillation (HD) HDL. See High-density lipoprotein (HDL) Health care products, 496. See also Skin care products; Therapeutic products compositions containing enriched natural crocin and/or crocetin, 496 drugs for kidney health, 496 hair conditioner, 496 toothpaste, 496 Healthy drink preparing from saffron pollen, 504
512 Heart rate (HR), 462 Hemagglutination (HA), 417 Hematocrit (HCT), 518 Hemoglobin (Hb), 518 Hepatic cancer, 489 Herba asari, 500 Herbal composition for treating diabetes, 498 Herbal medicine, 446, 517, 520 and AD, 449 formula for treating nasopharyngitis, 502 Herbicides, 175
176 Herniated disk, Chinese medicine for treating, 502 Herpes zoster treatment, 503
504 Hesperantha, 43 HI. See Harvest index (HI) High performance liquid chromatography
diode array detector (HPLC-DAD), 272
273 High pressure extraction (HPE), 270
272 High value agricultural product (HVAP), 219 High-density lipoprotein (HDL), 455, 471
472 High-density polyethylene, 303 High-performance liquid chromatography (HPLC), 273
279, 322, 324 High-performance liquid chromatographyultraviolet analysis (HPLC-UV analysis), 322 Hinduism, saffron color in, 16
17 Hippocampus, 431
432 Histamine, 408 History of saffron, 397 in traditional medicine, 394

537

usage in Persian civilization, 397 Honey suckle (Lonisera sp.), 496 Hordeum spontaneum, 174 Hormone application, 209 of saffron corm, 112 “Houli”, 18
19 HPA axis. See Hypothalamic-pituitary-adrenal axis (HPA axis) HPE. See High pressure extraction (HPE) HPLC. See High-performance liquid chromatography (HPLC) HPLC-DAD. See High performance liquid chromatography
diode array detector (HPLC-DAD) HPLC-UV analysis. See High-performance liquid chromatography-ultraviolet analysis (HPLC-UV analysis) HR. See Heart rate (HR) HTCC. See 4-Hydroxy-2,6,6-trimethyl-1cyclohexene-1-carboxaldehyde (HTCC) Hungry leeches (Chtonobdella limbata), 499
500 Hurdle technology effect on dried stigma microbiological quality, 316
317 HVAP. See High value agricultural product (HVAP) Hybrid photovoltaic
thermal solar dryer, 293
294, 293f Hydrastis canadensis. See Golden seal (Hydrastis canadensis) Hydrodistillation (HD), 266, 270 Hydroponics, 207
208 4-Hydroxy-2,6,6-trimethyl-1-cyclohexene-1carboxaldehyde (HTCC), 485
486 Hygienic postharvest conditions, 311, 312t Hygienic transportation, 311 Hyperglycemia, 424 Hypericum perforatum. See St. John’s wort (Hypericum perforatum) Hyperleptinemia, 472 Hypothalamic-pituitary-adrenal axis (HPA axis), 438 Hypothetical model of saffron development, 154
155, 154f

I ICAM-1. See Intercellular adhesion molecule-1 (ICAM-1) ICS. See Internal Control System (ICS) ICT. See Information and communication technologies (ICT) Ideal achievement, 346 IFN-γ/IL-4 ratio, 465 IFOAM. See International Federation of Organic Agriculture Movements (IFOAM) IgE. See Immunoglobulin E (IgE) Ikb kinase (IKK), 408
409 IL-1. See Interleukin-1 (IL-1) Ilex pubescens. See Maodongqing (Ilex pubescens) Immunoglobulin E (IgE), 408 Immunomodulatory effects of saffron

538

Index

Immunomodulatory effects of saffron (Continued) and derivatives, 412
417 derivatives, 415
417 extracts, 414
415 In vitro and preclinical studies, 423
427 AD, 426 memory and learning skills, 423
425 oxidative stress, 425
426 seizure, 427 In vitro cultivation, 208 In vitro research on saffron, 242t In vivo studies on effects of saffron and active compounds, 436
437 India, saffron production in, 341 Indian religions, saffron in, 16
19 celebrations and festivals, 18
19 color of religious costumes, 18 saffron color in Buddhism, 19 saffron color in India’s flag, 17
18 saffron for laundering gods, 17 symbols, 16
17 in Tantara rites, 17 Indigenous irrigation knowledge, 87
88 Indirect energy, 372 Induced nitric oxide synthase (iNOS), 408 Inflammation, 406 Information and communication technologies (ICT), 211 Informational function, 384 Infrared (IR) irradiation treatment, 314
315 radiation, 294 spectroscopy, 325
328 thin-layer drying, 294 Innovation in saffron production, 206
207 e-commerce, 212
213 forced flowering, 209 hormone application, 209 mechanization, 210
211 nonconventional breeding techniques, 209 organic production, 210 production in inadequate climates, 210 production under controlled environments, 207
208 saffron as feed additive, 211
212 saffron byproducts, 212 smart farming, 211 iNOS. See Induced nitric oxide synthase (iNOS) Input-based efficiency, 348 Insects (Insecta), 177
182 INSO. See Iranian National Standard Organization (INSO) Institute of Standards and Industrial Research of Iran (ISIRI), 315 Insulin-dependent diabetes. See Type-1 diabetes (T1D) Intercellular adhesion molecule-1 (ICAM-1), 408, 454 Intercropping, 109
110 Interleukin-1 (IL-1), 472 IL-1β, 410 Internal Control System (ICS), 210

International classification of saffron stigmas, 262 International Federation of Organic Agriculture Movements (IFOAM), 210 International Standardization Organization (ISO), 262, 368 Interspecific hybridization, 248 Intraperitoneally (IP), 452 Invented picker machines, 195
198 Invented separators, 200
203 IP. See Intraperitoneally (IP) IPP. See Isopentenyl diphosphate (IPP) IR injuries. See Ischemia-reperfusion injuries (IR injuries) Iran, 23, 292 saffron cultivation, 93 saffron production in, 340 use of saffron in ancient Iran, 25
28 Iran after Islam, saffron in, 28
32 applications in Iranian daily life, 30
32 dyeing, 30
31 food, 31
32 odor and aroma, 30 cultivation of saffron, 28 saffron as commodity, 28
30 Iran before Islam, saffron in, 23
28 Iranian National Standard Organization (INSO), 264 Iranian trade categories, 262
264 Iranian traditional medicine (TM), 393 Iridaceae, 41
43 Iris, 43 Irradiation treatment effect on dried stigma microbiological quality electron beam and gamma irradiation treatment, 314 microwave treatment, 313
314 Irrigation methods, 81
82 corm and stigma production of saffron, 82t scheduling before corm lifting, 70 after corm planting, 70, 71t preflowering irrigation in autumn, 71
72, 72t summer irrigation, 78
80, 79t during vegetative growth, 72
78 Ischemia-reperfusion injuries (IR injuries), 452 ISIRI. See Institute of Standards and Industrial Research of Iran (ISIRI) Islam, saffron in, 20
21 ISO. See International Standardization Organization (ISO) Isopentenyl diphosphate (IPP), 249 Isoprenoids. See Terpenoids Isoschizomers, 223

J Joe ghasem saffron (Crocus palasii), 52
53 Judaism, saffron in, 19
20 Juncus effusus. See Medulla junci (Juncus effusus)

K K-zeolite, 85
86 Kachi, 8 Kaempferol, 407
409, 409t, 436 Kalopanax septemlobus. See Acanthopanax (Kalopanax septemlobus) Karkom, 19
20 Kasmira. See Saffron (Crocus sativus L.) Ka´sm¯ırajanman. See Saffron (Crocus sativus L.) Kava kava (Piper methysticum), 497 Kerman province, 6
7 Kesar. See Saffron (Crocus sativus L.) Kesaravara. See Saffron (Crocus sativus L.) Keshmoon, 212
213 “Khoshki”, 6 Korkom, 24 Kowloon root (Ficus microcarpa), 499
500 Krocina tablet, 496
497 Kumkuma. See Saffron (Crocus sativus L.) Kurkum, 24

L Labor productivity of saffron, 345 Lactate dehydrogenase (LDH), 452
453, 518 Lactation day (LD), 520 LAI. See Leaf area index (LAI) LAMP. See Loop-mediated isothermal amplification (LAMP) Land productivity of saffron, 345 suitability for saffron, 161
163 LAR. See Leaf area ratio (LAR) Large gentian root (Gentiana lutea), 499 Large head atractylodes, 498 Laser-induced breakdown spectroscopy (LIBS), 328 LC-MS. See Liquid chromatography-mass spectrometry (LC-MS) LCA. See Lifecycle assessment (LCA) LCI analysis. See Lifecycle inventory analysis (LCI analysis) LD. See Lactation day (LD) LD50 value, 518 LDH. See Lactate dehydrogenase (LDH) LDL. See Low-density lipoprotein (LDL) LDL-C. See Low-density lipoproteincholesterol (LDL-C) LDPE. See Low-density polyethylene (LDPE) Leaf area index (LAI), 125
126, 126t, 150 Leaf area ratio (LAR), 131 Leaf weight ratio (LWR), 131
133 Learning skills, 423
425 Lepidium virginicum, 171
173 Leptin, 472 Leukemia, 488 LIBS. See Laser-induced breakdown spectroscopy (LIBS) Lifecycle assessment (LCA), 367
368 estimation of potential environmental impacts by, 367
372 AEP, 369
370, 370t AP, 369

Index

EcoX, 371
372, 371t GWP, 368
369 TEP, 370, 371t Lifecycle inventory analysis (LCI analysis), 368 Lifecycle of saffron, 122
123 dormant phase, 123 flowering phase, 122 production of replacement corms, 122
123 vegetative phase, 122 Ligusticum wallichii, 499 Limbic system, 431
432 Lindera aggeregata. See Spicebush (Lindera aggeregata) Linear low-density polyethylene (LLDPE), 303 Lipopolysaccharide (LPS), 407
408 Liquid chromatography-mass spectrometry (LC-MS), 325 Liquid cream, 495 Lithospermum arvense, 171
173 Liver cancer. See Hepatic cancer Liver damage, 479 LLDPE. See Linear low-density polyethylene (LLDPE) LOAEL. See Lowest observed adverse effect level (LOAEL) Long-term potentiation (LTP), 423 Longhairy antenoron herb (Lysimachia christiniae), 499 Lonisera sp. See Honey suckle (Lonisera sp.) Loop-mediated isothermal amplification (LAMP), 331 Low water availability, 85
86 Low-density lipoprotein (LDL), 471
472 Low-density lipoprotein-cholesterol (LDL-C), 455 Low-density polyethylene (LDPE), 303 Low-input agricultural systems, 373 Lowest observed adverse effect level (LOAEL), 519 LPS. See Lipopolysaccharide (LPS) LTP. See Long-term potentiation (LTP) Lung cancer/tumor, 486
487 Chinese medicine for treating, 502 Lung carcinoma. See Lung cancer/tumor LWR. See Leaf weight ratio (LWR) Lycium barbarum. See Wolfberry (Lycium barbarum) Lycopene β-cyclase (β-LCY), 250 Lyophilization. See Freeze drying Lyophilized saffron, 295 Lysimachia christiniae. See Longhairy antenoron herb (Lysimachia christiniae)

M m-chlorophenylpiperazine (mCPP), 437 MABP. See Mean arterial blood pressure (MABP) Maceration, 266
270, 267t Machines for corm production corm digging, 188
189 corm sorting, 189 physical properties of saffron corms, 188

Macrophage chemoattractant protein-1 (MCP1), 408 Macrophage inflammatory protein 2 (MIP-2), 408 Macroporous resins, 267t MADS-box proteins, 247 Major depressive disorder (MDD), 524
525 Malaytea scurfpea (Psoralea corylifolia), 498 Malondealdehyde (MDA), 452 “Mancha”. See Pooshal saffron “Mandala”, 17 Mantled trait, 222
223 Manure nitrogen content of, 64 to saffron, 62
64 Maodongqing (Ilex pubescens), 499 MAOIs. See Monoamine oxidase inhibitors (MAOIs) MAP. See Modified atmosphere packaging (MAP) MAPKs. See Mitogen-activated protein kinases (MAPKs) Marjoram (Origanum majorana), 79 Marketing of saffron, 357 concepts, 360
362 marketing management tasks for, 362
364 problems of exporting and, 357
360 Mashkzani, 9 Masks, 495
496 Matrix metalloproteinase (MMP), 486 MMP-2, 407
408 Maximal electroshock seizure (MES), 427 Maypop. See Passiflora incarnata 5-mC. See 5-Methylcytosine (5-mC) MCA. See Methylcholanthrene (MCA) MCDM. See Multi-criteria decision making (MCDM) MCDM
AHP method, 162 MCF-7 cells, 487
488 MCM. See Million cubic meters (MCM) MCP-1. See Macrophage chemoattractant protein-1 (MCP-1) mCPP. See m-chlorophenylpiperazine (mCPP) MDA. See Malondealdehyde (MDA) MDA-MB-231 breast cancer cells, 487
488 MDD. See Major depressive disorder (MDD) Meadow saffron flower, 3 Mean arterial blood pressure (MABP), 462 Measles, externally-applied wet tissue for treating, 503 Mechanistic models of plant growth, 139 simulation models, 148
149 Mechanization of saffron production, 210
211 corm planting, 191
194 economic advantages, 188 harvesting saffron flowers, 194
198, 196f machines for corm production, 188
189 role in agricultural development, 187 saffron stigma separation, 198
203 tillage, 189
191 Medicago lupilina, 171
173 Medication therapy, 433 Medulla junci (Juncus effusus), 500

539

Meiosis, 248 MEK-ERK1/2 pathway, 453 Melia azedarach. See Toosendan (Melia azedarach) Melting curve analysis, 329 Memantine, 446 Memory skills, 423
425 Meristem culture, 183 MES. See Maximal electroshock seizure (MES) Metabolic disorders, 471 effects of saffron on diabetes, 472
479 effects of saffron on obesity, 471
472 Methane (CH4), 368
369 Methyl orange, 322 Methylation-sensitive AFLP (MS-AFLP), 221, 222f, 226f Methylcholanthrene (MCA), 417 5-Methylcytosine (5-mC), 219
220 Mevalonate pathway (MEV), 249 Microbial decontamination of saffron, 310
315 Microbiological contamination of saffron, 313 Microbiological criteria of saffron antibacterial packaging effect on microbiological quality of dried stigma, 315
316 hurdle technology effect on microbiological quality of dried stigma, 316
317 microbial critical point in saffron, 308
309 microbial decontamination of saffron, 310
315 microbiological analysis classical culturing method, 308
309 rapid assessment by molecular analysis, 309 monitoring harvesting of saffron flowers, 309
310 saffron packaging, 315 transportation monitoring of saffron flowers, 310 Microcorm production, 235
236 Microextraction methods, 271 Microglial cells, 410 Microorganisms, saffron metabolites production in, 254
255 Micropropagation of saffron, 230
232 acclimation, 232 explant preparation, 230
231 propagation, 231
232 Microsimultaneous hydrodistillation
extraction (MSDE), 266 Microwave (MW) treatment, 313
314 Microwave drying, 295
296, 296f Microwave-assisted extraction, 267t Mid-infrared spectra (MIR spectra), 326
327 Million cubic meters (MCM), 206 Mini-mental state examination (MMSE), 445 MIP-2. See Macrophage inflammatory protein 2 (MIP-2) MIR spectra. See Mid-infrared spectra (MIR spectra) MIRI. See Myocardial damage after ischemia reperfusion (MIRI)

540

Index

Mite (Rhizoglyphus robini), 78, 177
182 distribution, 178 factors causing Rhizoglyphus robini to be problematic, 178 morphology and biology, 177 prevention and control in old farms, 179 prior to corms planting, 179
180 technical strategies to prevent and control saffron mites, 178 types of damage, 177
178 Mitogen-activated protein kinases (MAPKs), 407
408, 473
476, 519 Mix cropping, 174
175 MMP. See Matrix metalloproteinase (MMP) MMSE. See Mini-mental state examination (MMSE) Modeling saffron growth and development, advances in ANNs, 143
146 dynamic simulation models, 148
154 land suitability and zoning methodology for saffron, 161
163 modeling saffron development and flowering developmental responses in crop species, 155 hypothetical model of saffron development, 154
155 saffron response simulation to climate change, 158
161 structure of model, 155
158 response surface modeling, application of, 146
148 statistical models, 140
142 Modified atmosphere packaging (MAP), 303
304 Modified atmosphere packing, 315 Mofradat, 395 Molecular analysis, rapid assessment by, 309 Molecular biology of Saffron (Crocus sativus), 247 flower development, 247
248 gametogenesis and interspecific hybridization, 248 molecular response to abiotic stresses, 255 saffron metabolites production in microorganisms, 254
255 saffron
microbe interactions, 255 secondary metabolites, 248
254 Molecular techniques, 328
331 Monoamine, 434 Monoamine oxidase inhibitors (MAOIs), 432
433 Monocotyledons, tissue culture of, 230 Monoterpenes, 414 Moraea sisyrinchium, 173f Morakkabat, 395 Morus nigra. See Mulberry fruit (Morus nigra) Mother corms, 93
94 Motherwort (Nepeta cataria), 495 MPO. See Myeloperoxidase (MPO) MS. See Multiple sclerosis (MS) MS-AFLP. See Methylation-sensitive AFLP (MS-AFLP)

MSDE. See Microsimultaneous hydrodistillation
extraction (MSDE) Mulberry fruit (Morus nigra), 500 Multi-criteria decision making (MCDM), 162 Multicollinearity, 141 Multiglycosides saffron tablets, 498 Multilayer feedforward network, 143, 145 Multilayer perceptron (MLP). See Multilayer feedforward network Multiple regression models, 148
149 for saffron yield, 142, 142f Multiple sclerosis (MS), 426 Myeloperoxidase (MPO), 407
408 Myocardial damage after ischemia reperfusion (MIRI), 411 Myocardial ischemia, 452

N N-methyl D-aspartate (NMDA), 423
424, 438, 446 Najah, 4
5 Nanosilver composite antimicrobial packaging, 304 NAR. See Net assimilate rate (NAR) Nard (Nardostachys jatamansi), 499
500 extract, 424 Nardostachys jatamansi. See Nard (Nardostachys jatamansi) Nasopharyngitis, herbal medicine formula for treating, 502 National Control Drug Strategy, 342
343 Natural toxins, protective effects of saffron and active constituents on, 455
457 NCEDs. See 9-cis-Epoxycarotenoid dioxygenases (NCEDs) NE. See Norephinephrine (NE) Near-infrared spectroscopic imaging (NIR spectroscopic imaging), 325
326 Nematodes, 183 Nepeta cataria. See Motherwort (Nepeta cataria) Nervous system, 431
432 effect of saffron and compounds on diseases, 438
440 Nesokia indica. See Short-tailed bandicoot rat (Nesokia indica) Net assimilate rate (NAR), 130 Net energy, 378 Neurofibrillary tangles, 447
448 Neuroinflammation, 410 Neuroprotective agent, saffron application as AD, 445
446 herbal medicines and AD, 449 saffron in AD treatment, 446
448 NF-kB. See Nuclear factor-kappa B (NF-kB) NIK. See Nuclear factor-inducing kinase (NIK) NIR spectroscopic imaging. See Near-infrared spectroscopic imaging (NIR spectroscopic imaging) Nitric oxide (NO), 408, 453 Nitrogen (N), 62
63 requirement of saffron, 62
64 Nitrogen use efficiency (NUE), 64

in saffron, 64 Nitrous oxide (N2O), 368
369 NMDA. See N-methyl D-aspartate (NMDA) NMR spectroscopy. See Nuclear magnetic resonance spectroscopy (NMR spectroscopy) NO. See Nitric oxide (NO) NO observed adverse effect level (NOAEL), 519 Nominal Protection Coefficient (NPC), 351
352 Nominal protection coefficient inputs (NPCI), 352 Nominal protection coefficient outputs (NPCO), 352 Nonconventional breeding techniques, 209 Noninsulin-dependent diabetes. See Type-2 diabetes (T2D) Nonreproductive hybridization, 240 Nonulcer dyspepsia, Traditional Chinese medicine for treating, 503 Nopal (Opuntia sp.), 495 Norephinephrine (NE), 453 Normal drugs, 396 Normalized RMSE (NRMSE), 150 NORT. See Novel object recognition test (NORT) Notoginseng (Panax notoginseng), 499 Notopterygium (Notopterygium incisum), 499 Novel object recognition test (NORT), 424 NPC. See Nominal Protection Coefficient (NPC) NPCI. See Nominal protection coefficient inputs (NPCI) NPCO. See Nominal protection coefficient outputs (NPCO) NRMSE. See Normalized RMSE (NRMSE) Nuclear factor-inducing kinase (NIK), 408
409 Nuclear factor-kappa B (NF-kB), 407
408 Nuclear magnetic resonance spectroscopy (NMR spectroscopy), 328 NUE. See Nitrogen use efficiency (NUE) Nutrient pollution, 384 Nutrient removal, 62

O O-mics technologies for saffron valorization analysis of variation in saffron germplasm, 220
222 detection of adulteration and DNA-based traceability, 224
226 epigenetics stability, 222
224 perspectives, 226
227 Obesity effects of saffron on, 471
472 satiation agent for treatment of, 499 Obsessive-compulsive disorder (OCD), 435 OC. See Organic carbon (OC) Odor, saffron use as, 30 Oil-induced acne herbal soap, 495
496 Ointment for treating dermatitis, 497 Olea europea derivatives, 497

Index

Onion (Allium cepa), 50
51 Ophiocordyceps sinensis. See Tibetan worm grass (Ophiocordyceps sinensis) Optimizing models, 148 Opuntia sp. See Nopal (Opuntia sp.) Oral compositions, 497 Organic carbon (OC), 59 Organic fertilizers in saffron cultivation, 107
108 Organic production of saffron, 210 Organogenesis, direct, 233
236 generating adventitious shoots, 233
235 microcorm production, 235
236 Origanum majorana. See Marjoram (Origanum majorana) Osteoproliferation, Chinese medicine for treating, 502 Output-based efficiency, 348 Ovalbumin (OVA), 406, 464 ox-LDL. See Oxidized LDL (ox-LDL) Oxidative stress, 425
426, 447
448 Oxidized LDL (ox-LDL), 454 Oxygen, 301 production, 383 Ozone treatment effect on dried stigma microbiological quality, 313

P Packaging, saffron, 302, 315 aluminum foil, 302 edible and biodegradable films, 304 glass, 302
303 high-density polyethylene, 303 low-density polyethylene (LDPE), 303 modified atmosphere packaging (MAP), 303
304 modified atmosphere packing, 315 nanosilver composite antimicrobial packaging, 304 paper and paperboard, 302 Paeonia lactiflora. See White peony root (Paeonia lactiflora) PAM. See Policy matrix analysis (PAM) Panax ginseng root. See Ginkgo biloba leaf Panax notoginseng. See Notoginseng (Panax notoginseng) Pancreatic cancer, 487 Papaver rhoeas, 171
173 Paper and paperboard, 302 PAR. See Photosynthetically active radiation (PAR) Partial factor of productivity (PFP), 64 Partial nutrient balance (PNB), 64 Passiflora incarnata, 435 Passive cutaneous anaphylaxis (PCA), 408
409 Pathogens fungal agents of saffron corm rot, 182 Fusarium rottings of saffron corm, 182 nematodes, 183 saffron pathogenic bacteria, 183 saffron viruses, 183

PBMCs. See Peripheral blood mononuclear cells (PBMCs) PC-12. See Pheochromocytoma cells (PC-12) PCA. See Passive cutaneous anaphylaxis (PCA); Principal component analysis (PCA); Principal coordinate analysis (PCA) PCR. See Polymerase chain reaction (PCR) PDA. See Photodiode array (PDA) PDMS. See Polymethylsiloxane (PDMS) PDO. See Protected Destination of Origin (PDO) Peach (Prunus persica), 495
496 Penicillium digitatum, 102 Penman
Monteith method, 150 Pentylenetetrazole (PTZ), 427 Peptic ulcer, Chinese medicine for treating, 503 Perennials, 173 Peripheral blood mononuclear cells (PBMCs), 415 Peripheral nervous system (PNS), 431
432 Peroxisome proliferator-activated receptor α (PPARα), 455 Persian medicine (PM), 393 history of saffron usage in Persian civilization, 397 medical properties of saffron, 397
403, 399t botanical aspects, 397 medicinal uses of saffron, 402
403 nature and matter of saffron, 397
398 quality assessment, 403 temperament and general properties, 398
402 toxicity and adverse effects, 403 origin and history of saffron, 397 principles, 394
397 short history of using saffron in traditional medicine, 394 Persian miniature paintings, saffron in, 30 Persian religions, 15 Pests, 176
182 mites and insects, 177
182 harmful rodents in saffron fields, 180
182 Rhizoglyphus robini, 177
180 PFP. See Partial factor of productivity (PFP) PGE2. See Prostaglandin E2 (PGE2) PHA. See Phytohemagglutinin (PHA) Pharmaceutical industry, saffron in, 282
285 Pharmacotherapy, 395 Phenolic compounds, 249 Pheochromocytoma cells (PC-12), 447 Phonological description of saffron corm, 98 Photodiode array (PDA), 324 Photosynthetically active radiation (PAR), 153 Phylogeny, saffron, 43
46 Phytochemical analysis, 423 Phytoene biosynthesis, 253 Phytohemagglutinin (PHA), 415 Phytohormones, 209 Pichkari, 19 Picrocrocin, 252
253, 275t, 435, 485
486, 521

541

Piper methysticum. See Kava kava (Piper methysticum) Piper nigrum L. See Black pepper (Piper nigrum L.) PISTILLATA/GLOBOSA-like MADS-box genes, 247
248 PKC. See Protein kinase C (PKC) Plant cell, 229 Plant hormones, 112 Planting under controlled environment, 110
112 density, 104 patterns, 191
192 Plant organogenesis, 230 Platelets, 406 aggregation, 498 PM. See Persian medicine (PM) PNB. See Partial nutrient balance (PNB) PNS. See Peripheral nervous system (PNS) Poa bulbosa, 171
173 Policy analysis matrix, 349
351, 350t Policy matrix analysis (PAM), 382 Pollination of saffron, 46
47 Polygonum aviculare, 171
173 Polygonum multiflorum, 496, 500 Polymerase chain reaction (PCR), 309 amplification, 225f Polymethylsiloxane (PDMS), 271
272 Polynomial model, 147 Polyphenols, 414 Ponceau-4R, 322 Pooshal Negin saffron, 264 Pooshal saffron, 200, 200f, 264 Popular medicine, 8
9 Poria cocos, 500 Postharvest processes, microbial decontamination of saffron by, 310
315 cold plasma process effect on microbiological quality of dried stigma, 313 drying processes effect on microbiological quality of dried stigma, 311
312 IR irradiation treatment, 314
315 irradiation treatment effect on microbiological quality of dried stigma, 313
314 ozone treatment effect on microbiological quality of dried stigma, 313 Potential evapotranspiration, 67
70 PPARα. See Peroxisome proliferator-activated receptor α (PPARα) Precipitation, 121
122 Preflowering irrigation in autumn, 71
72, 72t corm and stigma production of saffron, 73t water availability effect on flower and stigma yield, 75t Pregnant women, saffron consumption by, 8 Premature ovarian failure, Chinese formulation for treating, 501 Prepregnancy fetus protection pills, 498 Price of saffron, 338
339 Primary hepatic cancer. See Hepatic cancer

542

Index

Principal component analysis (PCA), 279
281, 326
327 Principal coordinate analysis (PCA), 223f, 224 Product, price, place, and promotion (4 Ps), 362 Production of saffron Afghanistan, 342
343 under controlled environments, 207
208 growth chambers, 208 soilless beds, 207
208 in vitro cultivation, 208 efficiency, 346 function in economics, 343
344 of saffron, 344 Greece, 341
342 India, 341 Iran, 340 obstacles to, 205 Spain, 342 Programmed cell death, 486, 489
490 Programming languages, 149
150 PROMOTHEE method, 162 Propagation of saffron, 231
232 Prophylactic effect of saffron and derivatives on respiratory disorders, 464
467, 466t Prostaglandin E2 (PGE2), 408, 410 Prostate cancer, 490 Prostatic hyperplasia, medicine for treating, 500 Protected Destination of Origin (PDO), 282 Protein kinase C (PKC), 408, 490 Protein tyrosine phosphatase 1B, 473
476 Protoplast culture, 239
241 fusion, 240 Provisioning function, 381 Prunella vulgaris. See Spica prunellae (Prunella vulgaris) Prunus persica. See Peach (Prunus persica) Pseudomonas aeruginosa, 316 Pseudomonas gladioli. See Burkholderia gladioli Psoralea corylifolia. See Malaytea scurfpea (Psoralea corylifolia) Psychoanalytic theories, 433 PTZ. See Pentylenetetrazole (PTZ) Pubescent angelica (Angelica pubescens), 499 Pulsed electric field extraction, 267t Purple passionflower. See Passiflora incarnata Pyrrosia lingua, 500

Q Qanat system, 206 Qi stagnation, Traditional Chinese medicine preparation for treating, 502 Quality of saffron, environmental change effects on, 134
135 Quartet model, 247

R R-squared, 150 Radiation absorption, 153
154 Radiation use efficiency (RUE), 152

Radiation-based model for saffron growth, 152
154 inputs and parameters of model, 154 leaf growth and senescence, 153 radiation absorption and dry matter production, 153
154 structure, 153 Radio frequency, 292 Rainfed saffron production, 80 Raman spectroscopy, 328 Ranunculus arvensis, 171
173 RAP2.2 transcription factor., 253 Rapid assessment by molecular analysis, 309 RASFs. See Rheumatoid arthritis synovial fibroblasts (RASFs) Rate of return. See Return on investment (ROI) Ratio of return to cost (RRC), 346 RBC. See Red blood cell (RBC) RCA. See Revealed comparative advantage (RCA) RCWR. See Replacement corm weight ratio (RCWR) RDR. See Relative death rate of leaves (RDR) Reactive oxygen species (ROS), 415, 452, 489 Recurrent spontaneous abortion (RSA), 502 traditional Chinese medicine for treating blocking antibody deficiency, 502 Red blood cell (RBC), 518 Red gold. See Saffron (Crocus sativus L.) Reference evapotranspiration (ETo), 67
68, 152 Regression models, 141
144 Regulatory function, 381 Relative death rate of leaves (RDR), 153 Relative growth rate (RGR), 126
130 Relaxant effect of saffron derivatives on airway smooth muscle, 462
463 of saffron extracts on airway smooth muscle, 462 Renewable energy, 372 Renewable
nonrenewable energy ratio, 374 Replacement corm weight ratio (RCWR), 131
133 Replacement corms production, 122
123, 133 Resource use efficiency, 149 Respiratory system, saffron derivatives and effects on, 461 bronchodilatory effect of saffron and derivatives, 462
464 prophylactic effect of saffron and derivatives on respiratory disorders, 464
467 Response surface methodology (RSM), 146 application in saffron production, 147
148 CCDs, 146
147 statistical background, 146 Return on investment (ROI), 346 Revealed comparative advantage (RCA), 349 RGR. See Relative growth rate (RGR) Rheumatic arthritis Chinese medicine for treatment and prevention of, 499 topical treatment for, 499 Rheumatic heart disease, Traditional Chinese medicine for treating, 501

Rheumatism liquid patch, 497 Rheumatoid arthritis synovial fibroblasts (RASFs), 408 Rhizoglyphus robini. See Mite (Rhizoglyphus robini) Rhizoma alismatis (Alisma orientalis), 500 Rhizopus stolonifera, 102 Riddle, Saffron in, 9
10 RMSE. See Root mean square error (RMSE) ROI. See Return on investment (ROI) Romulea, 43 Root mean square error (RMSE), 142, 150 Root weight ratio (RWR), 131
133 ROS. See Reactive oxygen species (ROS) Rose mask, 495 RRC. See Ratio of return to cost (RRC) RSA. See Recurrent spontaneous abortion (RSA) RSM. See Response surface methodology (RSM) RUE. See Radiation use efficiency (RUE) RWR. See Root weight ratio (RWR)

S S-shaped function. See Sigmoid function (Sshaped function) SAE. See Saffron aqueous extract (SAE) Safety of saffron, 518
522 Safflower, 23
24, 499 Saffron (Crocus sativus L.), 3, 15, 23
24, 37, 41, 47f, 59, 93, 119, 140, 169, 219, 405, 423, 435
440, 451, 494, 517 antidepressant and anxiolytic properties, 437
438 bitterness, 282
283 byproducts, 212 C. hadriaticus, 54, 55f C. oreocreticus, 53
54, 54f C. palasii, 52
53 C. Thomasii, 54, 55f chemical compounds, 435 clinical studies on safe and toxic doses and bioactive ingredients, 523
526, 526t clinical trials on antidepressant and anxiolytic effect, 439t corms cultivation, 4 cultivated, 47
51 absorbing and contractile, 48f axillary buds, nodes, and internodes in saffron corm, 50f mother corm and new cormlets in saffron corm, 51f parts of saffron flower, 49f cultivation, 206 drying methods, 264, 265t effect and compounds on nervous system diseases, 438
440 evolution and botany contractile roots, 40
41 corm, 41 Crocus, 38
40 Iridaceae, 41
43 and phylogeny, 43
46 experimental data on safety and toxicity and bioactive ingredients, 518
522, 521t

Index

acute toxicity, 518 developmental toxicity, 520 mutagenicity and genotoxicity, 521
522 subacute toxicity, 518
519 subchronic and chronic toxicity, 519
520 folkore in planting, growing, and harvesting of, 4
5 and food, 5
6 germplasm, 220
222 mechanistic pathway for antidepressant and antianxiety effects, 438 metabolites production in microorganisms, 254
255 mycoflora, 183 pathogenic bacteria, 183 petals, 212 pharmaceutical applications, 436 pharmacology of saffron and active ingredients, 436 pollination and seed growth, 46
47 in popular literature, 9
11 Do-bayti, 9
10 riddles, 9
10 stories and humor, 10
11 popular medicine, 8
9 in prayers, charms, and talismans, 6
7 quality, 261 and religion, 15 in Indian religions, 16
19 in Semitic religions, 19
21 saffron-based compounds, 517 sexual reproduction, 46
47 uses of, 7
8 in vivo studies on effects and active compounds, 436
437 water, 7 wild, 51
52, 52f “Saffron All In One” (SAIO), 196, 197f Saffron aqueous extract (SAE), 490
491 Saffron marketing group (SMG), 363
364 Saffron stigma, 293
294 chemical analysis, 405 dehydration, 292 drying methods, 264, 265t international classification, 262 Saffron-based compositions, 499 Saffron
microbe interactions, 255 Saffrotin capsule, 500 Safra, 395 Safranal (SFR), 134, 252
253, 266, 412, 435, 447, 467, 473, 479, 485
486, 488, 520
521 determination, 275t experimental studies related to safety and toxicity, 524t immunomodulatory effects of, 417 mechanistic pathways for antidepressant effects, 440f Sage (Salvia miltiorrhiza), 495 SAIO. See “Saffron All In One” (SAIO) Salmonella spp., 308
309 S. enteritidis, 304 S. typhimurium LT2, 309 S. typhimurium tester strain TA98, 521

Salt stress in saffron, 83 Salvia chuanxiong (Salvia chuanxiensis), 500 Salvia miltiorrhiza. See Sage (Salvia miltiorrhiza) Salvia moorcroftiana, 171
173 Saposhnikovia divaricate. See Divaricate saposhnikovia (Saposhnikovia divaricate) SAPs. See Superabsorbent polymers (SAPs) Sargol Negin saffron, 264 Sargol saffron, 263
264 Sargols, 4
5 Satiation agent for treatment of obesity, 499 SBSE. See Stir-bar sorptive extraction (SBSE) SCAR. See Sequence-characterized amplified regions (SCAR) Scutellaria baicalensis. See Baikal skullcap root (Scutellaria baicalensis) Second-order drugs, 396 Secondary metabolites, 248
254, 297 apocarotenoid biosynthesis, 251
252 carotenoid biosynthesis in plants, 249
250 carotenoid biosynthesis in saffron, 251 crocin, crocetin, picrocrocin, and safranal, 252
253 genetic regulation of carotenoids biosynthesis, 253
254 Seed growth of saffron, 46
47 Seizure, 427 Selective serotonin reuptake inhibitors (SSRIs), 432
433 Semen strychni (Strychnos nux-vomica), 499 Semitic religions, 15
16 saffron in Islam, 20
21 Judaism, 19
20 SEPALIATA3 (SEP3)-like genes, 247
248 Septic shock, Chinese composition for treating, 504 Sequence-characterized amplified regions (SCAR), 329 Serotonin, 433 “Seven salams”, 7 Sexual reproduction of saffron, 46
47 SFE based on carbon dioxide (SFE-CO2), 270
271 SFE-CO2. See SFE based on carbon dioxide (SFE-CO2) SFR. See Safranal (SFR) “Shaeteh”, 25 Sheep red blood cells (SRBC), 414
415 “Shishtaki”, 4
5 Short-tailed bandicoot rat (Nesokia indica), 180 Sigmoid function (S-shaped function), 143 SigmStat package, 141 Signal transduction and activator of transcription 1 (STAT-1), 408 Silver ions (AgNO3), 210 Single nucleotide polymorphisms (SNPs), 221 Single-layer feedforward network, 143 Single-layer perceptron (SLP). See Single-layer feedforward network Sisyrinchium, 43 Skin cancer, 490

543

Skin care products, 494
496. See also Health care products; Therapeutic products creams, 494
495 masks, 495
496 Skin scars, topical spray for treating, 497 SLS. See Stigma-like structure (SLS) Smad3, 407
408 Smart Agricultural Knowledge and Innovation Systems Network (AKIS Network), 211 Smart farming, 211 SMG. See Saffron marketing group (SMG) SNPs. See Single nucleotide polymorphisms (SNPs) SOD. See Superoxide dismutase (SOD) Soda, 395 Soil conditions for sustainable saffron production, 60 nitrogen use efficiency in saffron, 64 saffron nitrogen requirement, 62
64 soil nutrient content, 61
62 soil texture, 60
61 Soil organic carbon, 59 Soil solarization, 174 Soilless beds, 207
208 hydroponics and aeroponics, 207
208 Solar power, 293 radiation, 149 Solar photovoltaic/thermal collector (Solar PV/ T collector), 293 Solid liquid extraction, 267t Solid-phase extraction, 267t Solid-phase microextraction (SPME), 266, 271 Somatic embryogenesis, 236
239, 238f Somatic embryos, 237
238, 240f Somatic hybridization, 240 Source and sink relationship in growth organs, 134 Southern mole vole (Ellobius fuscocapillus), 180 Soxhlet extraction, 270 Spain, saffron production in, 342 Sparganium stoloniferum. See Burreed tuber (Sparganium stoloniferum) Spatholobus suberectus stem, 500 Specific energy, 378
379 SPF. See Sun protection factor (SPF) Spica prunellae (Prunella vulgaris), 500 Spice, 517 trade, 321 Spicebush (Lindera aggeregata), 500 Spirit in PM, 397
398 SPME. See Solid-phase microextraction (SPME) SRBC. See Sheep red blood cells (SRBC) SSRIs. See Selective serotonin reuptake inhibitors (SSRIs) St. John’s wort (Hypericum perforatum), 497 Staphylococcus aureus, 308, 316 Star points. See Axial points STAT-1. See Signal transduction and activator of transcription 1 (STAT-1) Statistical models basics, 140

544

Index

Statistical models (Continued) crop-weather models for saffron yield prediction, 140
142 Statistical packages, 141 Stigma separation of saffron automated cutting machine, 202f correct cutting point, 201f invented separators, 200
203 physical properties of saffron flowers, 198, 200t saffron flowers sorter, 201f traditional stigma
flower separation method, 198
200 Stigma-like structure (SLS), 241
243, 241f Stir-bar sorptive extraction (SBSE), 271
272 Streptozotocin (STZ), 424, 455, 473, 476
477 Stress hormone, 432 Stress-inducible glycosyltransferase (CsGT), 255 Strychnos nux-vomica. See Semen strychni (Strychnos nux-vomica) STZ. See Streptozotocin (STZ) Summer irrigation of saffron, 78
80, 79t Sun protection factor (SPF), 284 Superabsorbent polymers (SAPs), 86 Supercritical CO2 extraction, 267t Supercritical fluid extraction, 270
271 Superoxide dismutase (SOD), 407
408, 452 Supervised learning, 143 Sustainable competitive advantage, 360 Synthetic polymer materials, 304

T T1D. See Type-1 diabetes (T1D) T2D. See Type-2 diabetes (T2D) Tang Loosha (ancient astronomical text), 27
28 Tantara rites, saffron in, 17 Tantara symbolism, 17 TaqMan polymerase, 309 “Tarhalva”, 5 Tartrazine, 322 Taste, effects of drying on, 296
297 Tau proteins, 447
448 TC. See Total cholesterol (TC) TCAs. See Tricyclic antidepressants (TCAs) TD. See Thermal desorption (TD) Tehran province, 6 Temperament, 394
395 of drugs and medicines, 396
397 and general properties, 398
402 Temperature, 119
121, 152, 158 Temporal lobe, 431
432 TEP. See Terrestrial eutrophication potential (TEP) Terpenes. See Terpenoids Terpenic aldehydes, 296 Terpenoids, 249 Terrestrial eutrophication potential (TEP), 370, 371t TG. See Triglyceride (TG) TGF-β1. See Transforming growth factor-β1 (TGF-β1)

Therapeutic products, 496
504. See also Skin care products; Health care products Thermal desorption (TD), 266 Thermal energy, 293
294 Thermotherapy, 183 Thin layer chromatographic method, 322 Third-order drugs, 396 Thr179, 407
408 Tibetan nighttime medicine, 497
498 Tibetan worm grass (Ophiocordyceps sinensis), 496 Tillage bed preparation for corm planting, 189
190 crust breaking, 190
191 Time series analysis of long-term yield data, 140 Tinglizi (Descurainia sophia), 171
173, 499 Tissue culture, 229 callus and cell culture, 232
233 direct organogenesis, 233
236 micropropagation of saffron, 230
232 of monocotyledons, 230 protoplast culture, 239
241 somatic embryogenesis, 236
239, 238f stigma-like structure (SLS), 241
243, 241f Tissue culture stigma. See Stigma-like structure (SLS) TLR4. See Toll-like receptor 4 (TLR4) TM. See Iranian traditional medicine (TM) TNBS-induced colitis. See 2,4,6Trinitrobenzene sulfonic acid-induced colitis (TNBS-induced colitis) TNF-α. See Tumor necrosis factor α (TNF-α) Toll-like receptor 4 (TLR4), 407
408 Toosendan (Melia azedarach), 501 Toothpaste, 496 Topical spray, 497 for treating skin scars, 497 Total cholesterol (TC), 455, 471
472 Total protein (TP), 406, 465, 467 Totipotency theory, 229 Toxicity of saffron, 518
522 TP. See Total protein (TP) Tracheal responsiveness, 406
407 Traditional Chinese medicine capable of treating cervical spondylosis, 501
502 composition for treating ascites due to cirrhosis, 503 preparation for treating qi stagnation and blood stasis, 502 for treating blocking antibody deficiency in RSA, 502 cardiovascular and cerebrovascular diseases, 504 gastric ulcer, 499
500 nonulcer dyspepsia, 503 rheumatic heart disease, 501 Traditional drying process, 311 Traditional planting methods, 192 Traditional stigma
flower separation method, 198
200 Transcription factors, 227

Transforming growth factor-β1 (TGF-β1), 407
408 Transmission FTIR spectroscopy, 327 Transportation monitoring of saffron flowers, 310 Tricyclic antidepressants (TCAs), 432 Triglyceride (TG), 455, 471
472 2,4,6-Trinitrobenzene sulfonic acid-induced colitis (TNBS-induced colitis), 411 Triticum aestivum. See Winter wheat (Triticum aestivum) True dormancy, 95 True passionflower. See Passiflora incarnata Tulipa stellata, 171
173 Tumor necrosis factor α (TNF-α), 410, 452
453, 472 Turmeric (Curcuma longa), 19, 23
24, 497
498, 500 Turnip (Brassica rapa subsp. rapa), 499
500 2K factorial points, 146 Type-1 diabetes (T1D), 472
473 Type-2 diabetes (T2D), 472
473

U UAE. See United Arab Emirates (UAE) UC. See Unit costs (UC) UDPG-glucosyltransferase (UGT), 252 UF. See Ultrafiltration (UF) UGT. See UDPG-glucosyltransferase (UGT) ULO. See Ultralow oxygen (ULO) Ultrafiltration (UF), 271 Ultralow oxygen (ULO), 208 Ultrasonic-assisted solvent extraction, 267t Ultrasound-assisted extraction, 267t Ultraviolet (UV), 302 sensitive products, 303 Unit costs (UC), 353 United Arab Emirates (UAE), 357 US Food and Drug Administration (FDA), 433 UV. See Ultraviolet (UV) UV-Vis spectrophotometry/spectroscopy, 272
273, 277t, 322

V V79 cells, 521 Vacuum collection, 198 V¯ahl¯aka. See Saffron (Crocus sativus L.) Validated regression model, 147 Valuation of ecosystem function, 382 Value-added variables, 142 Vascular cell adhesion molecule-1 (VCAM-1), 408 Vascular endothelial growth factor (VEGF), 408, 486 Vascular endothelial-cadherin expression, 411 Vascular smooth muscle cells (VSMCs), 454 Vasomodulatory effects of crocetin in hypertension, 453
454 VCAM-1. See Vascular cell adhesion molecule-1 (VCAM-1) Vegetable drinks, 504 Vegetative growth, 72
78

Index

Vegetative period of saffron, 119 VEGF. See Vascular endothelial growth factor (VEGF) Verdigris effects, 30 Very low-density lipoprotein-cholesterol (VLDL-C), 455 Vicia faba, 175 Vincristine, 487
488 Viruses, saffron, 183 Visual impairment, 284 VLDL-C. See Very low-density lipoproteincholesterol (VLDL-C) VSMCs. See Vascular smooth muscle cells (VSMCs)

W Warm and cold drugs, 396 saffron, 311 Water productivity (WP), 83
84, 151
152, 345 Water quality parameters, 82
83 salt stress in saffron, 83 Water requirements of saffron, 67
68 beneficial water-related approaches for saffron production, 85
87 crop coefficients and potential evapotranspiration, 67
70 factors affecting water requirements, 80
81 indigenous irrigation knowledge, 87
88 irrigation methods, 81
82 irrigation scheduling, 70
80

physiological responses of saffron to water stress, 84
85 rainfed saffron production, 80 saffron response to flooding, 87 water quality, 82
83 water-use efficiency and productivity, 83
84 Water stress, physiological responses of saffron to, 84
85 Water-use efficiency, 82
84, 87t WBCs. See White blood cells (WBCs) Weak stigma, 262 Weeds, 169
176 critical period of weed control, 171 dominant weeds in saffron fields, 171
173 intervention, 169 management, 174
176 chemical control, 175
176 cover crops, 175 mechanical control, 174 mix cropping, 174
175 monitoring, 174 prevention, 174 soil solarization, 174 presence in saffron fields and possibility of weed control, 170
171 and saffron ecophysiology, 170, 172t Wheat residue, 109 White blood cells (WBCs), 406, 464, 518 White monkshood (Aconitum napellus), 495 White peony root (Paeonia lactiflora), 495 WHO. See World Health Organization (WHO) Wild apricot. See Passiflora incarnata

545

Wild passion vine. See Passiflora incarnata Wild rue, 4 Wild saffron (Crocus cartwrightianus), 51
52, 52f Winter wheat (Triticum aestivum), 155 Wolfberry (Lycium barbarum), 498 World Health Organization (WHO), 393, 471 World Saffron and Crocus Collection (WSCC), 220 accessions, 220t, 221 WP. See Water productivity (WP) WSCC. See World Saffron and Crocus Collection (WSCC)

X Xenopus embryos, 520

Y “Yantra”, 17 Yemeni saffron, 19
20

Z Zedoary (Curcuma zedoaria), 500 ζ-carotene isomerase (Z-ISO), 250 Zingiber officinale. See Ginger (Zingiber officinale) “Ziphron”, 20 Zirehjoosh, 6 Zoning methodology for saffron application, 162
163 objectives and methods, 161
162